The photosensitizer and method thereof

 

(57) Abstract:

The invention relates to medicine and relates to a photosensitizer for photodynamic therapy and method of its production, including alkaline salt of chlorin e6, purpurine 5 and chlorin p6characterized the optimal rate of accumulation in the tumor and its removal of the tumor, as well as high stability. 2 C. and 7 C.p. f-crystals, 18 ill., 3 table.

The invention relates to the chemistry of biologically active compounds in the field of photodynamic therapy (PDT).

Photosensitizers (PS) are used in photodynamic therapy as therapeutic agents and photodynamic diagnosis (PDD) as fluorescent labels.

Known FS Terentieva salt of mono-L-aspartyl chlorin e6"Npe6" [1]:

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This FS has activity during PDT.

Its disadvantages are the greater complexity of obtaining excessively accelerated dynamics of accumulation in the tumor and removing it, which reduces the time for effective action on the tumor, as well as a relatively low degree of accumulation in malignant tumors due to the significant hydrophilicity, which uses only one of the few who CLASS="ptx2">

Known FS trinacria salt lysyl-chlorin p6"LCP" [2]:

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This FS has pronounced activity during PDT.

Its disadvantage is the high complexity of receipt, and that it is a mixture of monoamines to 13 and 15 to the provisions in the ratio of about 10: 1, which may lead to ambiguous biodistribution and excretion.

Known FS - sodium salt of pheophorbide a [3]:

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This FS has the ability to selectively accumulate in malignant tumors and activity during PDT.

Its disadvantages are high propensity for oxidation (chemical instability) during storage in solution, incomplete solubility after storage in solid form, hydrophobicity and, consequently, slow excretion from the body, which leads to prolonged photosensitivity of the skin.

Known FS - derivative of chlorin e6[4]:

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where R = hydrophobic hydrocarbon Deputy, saturated or unsaturated, straight or branched, containing from 4 to 25 carbon atoms.

FS, in which R = hexyl, has trapnest against malignant tumors and is an effective means is wah, high hydrophobicity and, consequently, a slow accumulation in the tumor and low stability of aqueous solutions of dosage forms during storage (prototype).

A method of obtaining FS, namely the composition of the chlorines in the form of salts with alkali metal, intended for medical use, namely, that the plant biomass is extracted with a mixture of hydrocarbons containing 6 to 12 carbon atoms, and alcohols containing 2-10 carbon atoms, taken in a volumetric ratio of from 2:1 to 8:1, the resulting solution was evaporated chlorophylls at atmospheric pressure, add alcohol containing fewer carbon atoms than taken for extraction, completely distilled off from the mixture of hydrocarbons at atmospheric pressure, gradually added to the alcohol solution of chlorophylls alcoholic alkali solution at the boiling temperature of the alcohol, but less than 120oWith, to a pH of 11.5 and 11.8, cool, soak 4 hours, filtered, extracted with a mixture of hydrocarbons containing 6 to 12 carbon atoms, separating the alcohol phase containing magnesium complexes of chlorine, evaporated alcohol at atmospheric pressure, is added to the residue hydrochloric acid to pH 3.5, stand before the termination of the precipitation chlorine is a thief FS and it evaporated in vacuum [5].

The disadvantage of this method is the use of high temperatures during the removal of solvent from the extract, the use of alcohols, particularly methyl, leading to allometrically of eksotika E and education from pheophytin and pheophorbide a large number of various oxidation products [6] , which leads to a complex mixture of uncertain and difficult reproducible composition.

A method of obtaining FS, namely sodium salt of chlorin e6according to which 1N NaOH solution was added to a solution of trimethylboron ether of chlorin e6in tetrahydrofuran, and then the reaction mass is stirred for 2 days at room temperature in a nitrogen atmosphere and add water, then the organic solvent is extracted with methylene chloride, removing the last traces by bubbling nitrogen through the solution of salt of chlorin e6[4].

The disadvantages of this method are the inaccessibility of significant quantities of source trimethylboron ether of chlorin e6the duration of the process of obtaining from him the FS due to the chemical inertness of the ester residue in the 13th position of the tetrapyrrole macrocycle and the instability of the dosage forms FS when stored in the form of water reposal receiving FS, namely, a photosensitizer for photodynamic therapy "LCP" (trinitarios salt lysyl-chlorin p6), namely, that process the biomass 2-3 times with acetone for the extraction of chlorophyll and sucked biomass or centrifuged her, evaporated extract, process, extract acid for removal of chlorophyll molecules of the magnesium ion and hydrolysis failboy broadcasting group with the addition of methyl alcohol for simultaneous esterification process of the reaction mass with water, extracted with derived pheophorbide and methylene chloride, the extract is neutralized, washed with water, evaporated, chromatographic on aluminium oxide, methylpheophorbide and crystallized from a mixture of methylene chloride-methanol and injected derived derived pheophorbide and reaction with a strong inorganic base in the presence of oxygen in pyridine - diethyl ether - n-propanol, treated with the reaction mass with water, the aqueous phase is acidified to pH 4, extracted with unstable chlorin" methylene chloride, evaporated extract, pererastayut unstable chlorin" in tetrahydrofuran, evaporated solution, repeat until the termination of growth of the absorption at 700 nm, dissolved received purpurine 18 in tetrahydrofuran, Etna in the presence of pyridine, stir the mixture for 12 hours at room temperature, remove the solvent in high vacuum, the resulting crude product is purified using reverse-phase HPLC, remove the solvents lyophilic drying, dissolved FS in phosphate buffer with the aim of obtaining injectable solution for PDT, add 0,1 N NaOH solution, bring the solution to a physiological pH of 7.35 using a 0.1 N HC1 and filtered through a microporous filter [2] (the prototype).

The disadvantages of this method include poor reproducibility, the complexity (use of high vacuum, crystallization, column chromatography and HPLC, long reaction with lysine), the use of highly toxic and flammable reagents (diazomethane, pyridine, methanol, tetrahydrofuran, diatrofi ether), making it unsuitable for pharmaceutical production. In addition, the obtained water-soluble target product is stable in aqueous solution only 24 hours at 4oIn the dark and in solid form only up to 4 months at 4oIn the dark, as necessary, in accordance with the requirements of the Pharmacopoeia, not less than 6 months [7]. Moreover, from the point of view of chemistry, this FS is a mixture of monoamide 13 and 15 the provisions of the Oia from the body.

The present invention is FS, which is characterized by ease of isolation and purification in preparative quantities, balanced hydrophobicity-hydrophilicity and, as a consequence, the optimal rate of accumulation in the tumor and excretion (from the tumor and from the body as a whole), as well as high stability of aqueous solutions of dosage forms during storage.

This problem was solved by creating a filesystem containing chlorine in the form of a salt with an alkaline metal, and as chlorine is taken chlorin e6(13-carboxy-17-[2-carboxyethyl] -15-carboxymethyl-17,18 - TRANS-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylpiperidine)

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in the amount of 80-90%, purpurine 5 (13-carboxy-17-[2-carboxyethyl]-15-formyl-17,18-TRANS-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylpiperidine)

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in the amount of 5-20%, and purpurine 18 - chlorin p6(13-carboxy-17-[2-carboxyethyl] -15-carboxy-17,18-TRANS-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylpiperidine)

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the amount of rest so that these components constitute the song, and as the alkali metal can be used sodium or potassium.

The present invention is also an achievement in the way of getting FS high vosproizvodimosti 1 year the combination of physico-chemical and biological properties of FS that will ensure its effectiveness in PDT.

The method of obtaining the FS is that process the biomass of Spirulina acetone until complete extraction of chlorophyll a, filtered biomass or centrifuged her, treat the extract with acid to remove the chlorophyll molecules of the magnesium ion, neutralize the extract is precipitated and filtered pheophytin a, then hydrolyzing pheophytin and in a mixture of hydrochloric acid-acetone-hexane, with each 1 g of the crude pheophytin and take 6-16 ml of acetone, 0.6 to 6 ml of hexane and 5-10 ml of concentrated hydrochloric acid, heat the mixture to a temperature of 40-60oC and stirred for 20 min - 1 hour, then add hexane (6-16 ml) and the organic phase is washed with a mixture of acetone and concentrated hydrochloric acid (2-10):1, the aqueous phase is washed with hexane, then neutralized aqueous phase containing pheophorbide and an excess of an aqueous solution of sodium citrate (three-, two - or one-deputizing), separating the rolled deformed but by filtration, washed with water, periostat it from a mixture of acetone-water, air-dried until constant weight, then dissolve pheophorbide and in acetone, add strong reorganizes billaut additional amount of a strong inorganic base in aqueous solution with a concentration of 1-50%, heated at 40-60oFor 20-90 min, neutralized mixture of diluted hydrochloric acid, separating the precipitate of chlorin e6by centrifugation, washed it with distilled water until the disappearance of the acid reaction, get 55-80% of chlorin e6, periostat chlorin e6from acetone to separate linear tetrapyrroles, filtered chlorin e6and washed it with distilled water, heated chlorin e6in sealed containers within the temperature range 40-100oC for 1 hour to 30 hours, cool and add a solution of a strong base to a pH of 7.5-8.5 and Ousterhout pyrogen-free water for injection to the content of the photosensitizer of 6.5-7.5 wt.%.

In addition, in the method of obtaining FS after you add a solution of a strong base to a pH of 7.5-8.5, the mixture can be subjected to gel filtration to the content of chlorin e6- 80-90%, purpurine 5 - 5-20% and purpurine 18 - the rest, then added a dilute solution of hydrochloric acid and the precipitate discarded photosensitizer, are now aligned with pyrogen-free water for injection to the content of the photosensitizer of 6.5-7.5 wt. % and the resulting "Liquid extract of chlorins".

In addition, in the method of obtaining the FA, after gel filtration to restoreselection, the residue is filtered or separated by centrifugation, added allowed by the State Pharmacopoeia of the Russian Federation supplements to pH 7.5-8.5 and pyrogen-free water for injection to the content of the photosensitizer is 0.1-1 wt.% and filtered from bacteria.

In addition, in the method of obtaining FS after gel filtration the mixture may be added a dilute solution of hydrochloric acid until precipitation of the photosensitizer, the residue is filtered or separated by centrifugation, adjusted pyrogen-free water for injection to the content of the photosensitizer of 6.5-7.5 wt.%, dispersed "Liquid extract of chlorins" in gel base rate of 0.5 to 12 wt.% "Liquid extract of chlorins", 5-20 wt.% dimethyl sulfoxide and the rest is water, is allowed by the State Pharmacopoeia of the Russian Federation supplements and gel-based.

In addition, in the method of obtaining FS after gel filtration the mixture may be added a dilute solution of hydrochloric acid until precipitation of the photosensitizer, the residue is filtered or separated by centrifugation, adjusted pyrogen-free water for injection to the content of the photosensitizer of 6.5-7.5 wt.%, and the resulting "Liquid extract of chlorins" dissolved in dimethyl sulfoxide based: 0.5 to 12C by using standard laboratory chemical pilot equipment: process the biomass in aluminum cans with a capacity of 10-50 l, equipped with a mechanical stirrer, filtered biomass by suction filters with a capacity of 5-20 l vacuum oil pump and cooled by a liquid nitrogen trap, centrifuged biomass using floor centrifuge with glasses 4x1 l with cooling and speed up to 6000 rpm, process, extract acid in glass bottles with a capacity of 20 liters, filtered fallen pheophytin and suction filters with a capacity of 5-10 l vacuum oil pump and cooled by a liquid nitrogen trap, hydrolyzing pheophytin and in a three-neck round-bottom flasks with a capacity of 0.1-0.5 l, equipped with heating, mechanical agitation, reflux condenser and an inlet opening from the tube, washed with solutions using separating funnels with a capacity of 2 l, neutralized in glasses chemical capacity 2-5 l, filter pheophorbide and suction filters with a capacity of 2-5 l vacuum oil pump and cooled by a liquid nitrogen trap, periostat in flat-bottomed flasks chemical capacity of 0.25-1 l, dissolve pheophorbide and in acetone and added a strong inorganic base in a three-neck round-bottom flasks with a capacity of 0.5-2 l, equipped with heating, mechanical stirring, reflux condenser and zagrai centrifuge with glasses 4x0,5 l with cooling and speed up to 6000 rpm, periostat chlorin e6using a flat-bottomed flasks chemical capacity of 0.25-0.5 l 2-5 l, filtered chlorin e6and by suction filters with a capacity of 1-2 l vacuum oil pump and cooled by a liquid nitrogen trap, heated chlorin e6in chemical round-bottom flasks from heat-resistant glass with a capacity of 0.05-0.1 l, add a solution of a strong base and Ousterhout in chemical glasses with a capacity of 0.1 to 1 l using a standard pH meter and spectrophotometer, the mixture is subjected to gel filtration on a column with a diameter of 50-100 mm and a height of 100-150 mm, filtered from bacteria using standard Millipore Millipore filters with a pore diameter of 0.22 μm, is dispersed Liquid extract of chlorins" in gel base using a knife or ball homogenizer, in addition, for the preparation of portions, solutions and samples used conical flasks with stoppers capacity of from 0.01 to 10 l, cylinders with a capacity of 0.005 to 2 l, glasses with a capacity of from 0.05 to 2 litre bottles with a capacity of 20 l, scales weighing range 1-1000 g, magnetic stirrer; for the regeneration of acetone and hexane - neck round bottom flask with a capacity of 5 l with a thermometer and once-through water fridge for quick removal rastvoritelyami hydrochloric acid is considered a saturated solution of hydrogen chloride in water at a temperature of 20oWith that commonly contains 36-37 wt.% of hydrogen chloride.

When turning pheophytin and pheophorbide and the range of quantities of hexane and acetone (6-16 ml of acetone and 0.6 to 6 ml of hexane) is related to the fact that fewer solvents and pheophytin dissolved not completely, but at a higher - gain solution concentrated enough for a quick course of hydrolysis. The range of quantities of hydrochloric acid (5-10 ml) is related to the fact that fewer of hydrochloric acid decreases the output pheophorbide a, but at a higher - selectivity of the reaction, as it produces a lot of side product pyropheophorbide and. The range of temperatures of 40-60oWith related to the fact that at lower temperature decreases the output pheophorbide and, in higher decreases the selectivity of the reaction due to the formation of side product pyropheophorbide and. The range of values of the reaction time of 20 min - 1 hour - is that with less time decreases the output pheophorbide and, in higher decreases the selectivity of the reaction due to the formation of side product pyropheophorbide and. The amount to be added next hexane - 6-16 ml - due to the fact that when the smaller number from the reaction mass not is consistent.

When cleaning pheophorbide and the organic phase is washed with a mixture of acetone and concentrated hydrochloric acid, taken in the ratio from 2:1 to 10:1. At a ratio less than 2:1 mixture of outstanding flocculent precipitate impurities, which is poorly separated from the aqueous phase containing the target pheophorbide and. When the ratio is about 10:1 aqueous phase presidets acetone, and move impurities from the hexane phase, polluting target pheophorbide A.

When turning pheophorbide and chlorin e6the concentration of a strong base set in the range of 0.05 to 1.00%, and the lower bound is the minimum needed for reaction disclosure Cyclopentanone ring (ring E) pheophorbide and a when the alkali concentration greater than 1% of the reaction takes place allometrically (oxidation) of the E ring, which leads instead of the target of chlorin e6to "unstable chlorin", and then to purpurine 18 and further to the chlorin p6.

Further in the method adds an additional amount of a strong inorganic base in aqueous solution with a concentration of 1-50%. When the alkali concentration of less than 1% is incomplete saponification of the ester residue 13 and/or 15-position. When using alkali in a concentration of more than 50% of therapyoC for 5-30 min, and a lower temperature facilitates the process of allometrically the E ring, and large - decomposition of chlorin e6to chlorin e4. Less time the process is not sufficient for reaction to the disclosure of the E ring, and a larger increases the output side of chlorin e4. When adding additional amounts of strong inorganic bases temperature range - 40 to 60oC, and reaction time - 20-90 minutes At a lower temperature values and time does not have time to either hydrolyzed methyl ester at position 152when large increases output side of chlorin e4.

When the transformation of chlorin e6in "Liquid extract of chlorins" the process of oxidation and subsequent thermolytic processes of dehydration and decarboxylation of PS with oxidized methylene group in position 151in purpurine 5:

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When the transformation of chlorin e6in "Liquid extract of chlorins" using temperature below 40oWith requires a long time process that is technologically justified. The use of temperatures above 100oWith leads to the accelerated decomposition of the substance.

Of provia, with low biological activity.

The process is more than 30 days accompanied by irreversible change (decay) of a substance.

The optimal temperature of the process is 45-70oWith (Fig. 1).

The best time of the process are 2-9 days at 70oWith (Fig. 2) or 1-48 hours at 100oWith (Fig. 3), resulting in 5-20% of purpurine 5 in the mix.

Substance containing 5-20% of purpurine 5 and 80-95% of chlorin e6part of the active start (FS), suitable for water-soluble injectable dosage forms. If the substance contains less than 5% of purpurine 5, it has a low biological activity. If the substance contains more than 20% of purpurine 5, its solubility in water is deteriorating, that adversely affects the stability of the dosage forms during storage and impairs the ability to filter through Millipore filters. The latter property is necessary for sterilization dosage forms, as dosage forms tetrapyrroles cannot be sterilized by heat or UV rays due to the high probability of chemical reactions.

The presence of the substance 80-95% of chlorin e6need is oflen fact, that the bottom boundary - pH 7.5 - is the lower limit of the solubility of chlorine in aqueous solutions to produce concentrations suitable for use in the pharmaceutical industry, without adding solubilization. The upper limit of this range is a pH of 8.5 is a biological limit portability concentrations of hydroxide ions, [HE-].

The concentration range of chlorin e6of 6.5-7.5% due to the use of technological methods of centrifugation or filtration stage precipitate of chlorin e6giving the product in this range of concentrations.

The invention is illustrated by drawings, in which Fig. 1 relates to a method and shows the formation of purpurine 5 depending on the temperature during curing for 30 days; Fig. 2 shows the dependence of the content of purpurine 5 from the time-keeping at a temperature of 70oC; Fig. 3 shows the dependence of the content of purpurine 5 from the time-keeping at a temperature of 100oC; Fig. 4 illustrates the pharmacokinetics of the substance "Liquid extract of chlorins", used in the form of dosage forms "Radachlorin, 0.5% solution for injections" ("Potohari") in tumor mice when administered intravenously at a dose of 20 mg/kg; Fig. 5A PE, designated as "1", cleared for FS in 0.01 M borate buffer with a pH 9,18 and the curves marked as "2", filmed for FS in blood; Fig. 5B confirms the metabolism of chlorin e6(formula I) in purpurine 5 (formula II) in the liver; Fig. 6 depicts the PMR spectrum of the substance "Liquid extract of chlorins" obtained in example 2; Fig. 7 shows the mass spectrum of the substance "Liquid extract of chlorins" obtained in example 2; Fig. 8 gives the absorption spectrum in the visible region of the substance "Liquid extract of chlorins" obtained in example 2, the spectrum is cleared in ethanol for 5 µg/ml of the substance; Fig. 9 presents the PMR spectrum of chlorin e6; Fig. 10 contains the mass spectrum of chlorin e6; Fig. 11 shows the absorption spectrum in the visible region of chlorin e6spectrum is cleared in ethanol and conc. chlorin e6, 15 µg/ml (lane Sora - 5 µg/ml); Fig. 12 shows the PMR spectrum of purpurine 5; Fig. 13 provides the mass spectrum of purpurine 5; Fig. 14 contains the absorption spectrum in the visible region of purpurine 5; range is cleared in ethanol and conc. of purpurine 5-15 µg/ml (lane Sora - 5 µg/ml) Fig. 15 gives the PMR spectrum of the dimethyl ether of purpurine 5; Fig. 16 presents the mass spectrum of the dimethyl ether of purpurine 5; Fig. 17 Dan absorption spectrum in the visible region of dimethyl ether purpurine what iMER 1, examples of the method are given in examples 2, 3, particular cases of the implementation of the method illustrated in examples 4-9.

FS from the point of view of chemistry contains three circular tetrapyrrole chlorin nature (gidrirovanny ring D) - chlorin e6(formula I), purpurine 5 (formula II, example 10) and purpurine 18, which in alkaline medium (storage) gradually turns into a chlorin p6(formula III).

FS from the point of view of physical chemistry has the ability to absorb light in the visible region, resulting in the photo-activation and subsequent relaxation of the excited state by energy transfer to dissolved in the tissues of molecular oxygen and organic substrates. The latter leads to oxidative and free-radical processes in biological tissues and their damage and subsequent destruction (necrosis). Most preferred for PDT band excitation is the long wavelength band (PL. 1) because with increasing wavelength increases the penetrating power of light in biological tissue. Thus, the FA can destroy biological objects after excitation with light of wavelength 654-670 nm at a depth of 10 mm

FS from the point of view of pharmaceuticals is the company's less than 20%). Extract this substance is due to the necessity of extraction of the biomass with the use of organic solvents.

The compound of formula II has the ability to selectively accumulate in malignant tumors and infected lesions, but poorly soluble in water, and the compound of formula I, along with a strong photodynamic activity is solubilizers means for compounds of formula II.

From the point of view of pharmacology (Fig. 4, example 11), the uniqueness of pharmacokinetic parameters is achieved by the fact that FS formula (I) in the body slowly turns into FS formula (II), which supports the concentration of the latter at a constant level since the introduction into the body until the removal of the tumor, within a period of time sufficient for effective PDT. After injection into mice-carriers of tumour proposed composition of the Federal Assembly, it gets into the bloodstream and through the circulation in the blood primarily compounds of formula (I) in the first 3 hours after administration in the area of the tumor is high and stable concentration of FS - 0,27-0,32 M, sufficient for effective PDT in the range of 0.5 to 4 hours. High kontrastnosti has the property of high contrast to accumulate in the tumor, moreover, the maximum accumulation accounts for 3 hours after administration of the Fund in the animal organism (the index contrast is 14.5 skin and 2.9 muscles). During this time, the compound of formula (I) is converted in the body into the compound of the formula (II), providing a stable high concentration of FCS in the area of the tumor in the range of 3-5 hours after injection, which is gradually decreasing, remaining therapeutically sufficient up to 18 hours after injection. The compound of formula (II) further breaks down in the body to non-toxic products, which are excreted by the liver.

The transformation of chlorin e6formula (I) in purpurine 5 of the formula (II) is confirmed by the fluorescence spectra of samples of organs and tissues of experimental animals (Fig. 5). Adding dosage forms "Radachlorin, 0.5% solution for injections" ("Potohari") to the homogenate blood [Fig. 5A, (1)] at a concentration of 10 M, followed by a spectrophotometric study observed the change in the spectrum of fluorescence in the form of broadening by a factor of 1.2 and a shift of the maximum of fluorescence intensity in the long wavelength part of the spectrum of 8 nm. Adding "Photochlorin" in lower concentrations (=1 M) were observed shift of the spectrum without broadening, which demonstrates the effect dosta blood, obtained 3 hours after injection "Photochlorin" mice. Here is most pronounced measure of the broadening of the spectrum at 1.5 times maximum fringe shift in the long wavelength part of the spectrum of 4 nm, which implies that in the sample of blood is a mixture of "Photochlorin" and metabolite.

Adding "Photochlorin" in the tube with the homogenate of the liver [Fig. 5B (1)] at a concentration of 10 M, the change in the spectral characteristics are expressed primarily in the shift of the maximum of fluorescence intensity in the long wavelength part of the spectrum to 9 nm. The broadening of the spectrum is missing.

A similar pattern is observed when adding "Photochlorin" in lower concentrations C=1 M [Fig. 5B, (2)].

When studying homogenate of liver tissue obtained 3 hours after injection the animals, spectrophotometric picture of the sample is similar to the previous two [Fig. 5B (3)].

Thus, the obtained data demonstrate the presence of metabolite "Photochlorin" in the homogenates of the liver.

Optimal selectivity for photodynamic accumulation of purpurine 5 in tumors in experimental animals observed in the range of 3-18 hours after intravenous or Nutripro the current the optimal exposure time is 0.5 to 4 hours after intravenous injection. In General, for PDT with the substance of "Liquid extract of chlorins" the interval between administration of the drug and the dose is 0.5 to 18 hours.

Biological activity of medicinal forms "Radachlorin, 0.5% solution for injections" ("Potohari") containing 0.5% anhydrous substance "Liquid extract of chlorins", evaluate in vitro and in vivo.

The balance of the Federal Assembly of amphiphiles confirm the standard in vitro experiment [8] (table. 2, example 12). The distribution coefficient FS in 1-octanol/phosphate buffer, pH 7.4 (Kp), equal to 1.40. This means that the proposed FS equally well soluble both in aqueous and the lipid phase and proves the lipophilicity of the Federal Assembly, which allows this connection to be transferred from the water in the complexes with transport proteins and lipoproteins, quickly penetrate into the cells and accumulate in cytoplasmic intracellular membranes and microsomes, or you can enter cells by diffusion through the plasma membrane of these cells. After laser irradiation, thus deposited connection allocates singlet oxygen inside the cell, killing it.

the tatami obtained in vitro, in which he used 3 lines of cultured tumor cells of rat pheochromocytoma PC 12, neurinomas Gusarova node rats NGOK and rat hepatoma 27 (Ner) (table. 2, example 13).

For research dasavasebeli cytopathologically (after laser exposure and biological "dark" activity FS use the following methods:

1. MTT-test, which allows to accurately determine the number of living cells after they are processed FS and irradiation with a laser to calculate the cytotoxic and cytopathological indexes FS. This test allows to evaluate gotovisya cytotoxic and biological tenovuo activity FS [9].

2. Determining the number of cells after staining the cell monolayer crystal-Violeta at the end of the experiment. This method is less time-consuming and expensive than the MTT-test also allows us to calculate the cytotoxic and cytophotometrically indexes FS [10], but it is less accurate, because the crystal violet stain and dead cells.

3. Comparative genotoxic and geopoliticheskoe action FS assess the degree of inhibition of DNA synthesis in cells. The amount of DNA synthesis determined by the level of incorporation into DNA14With-thymidine, COI is in the action of laser irradiation after they are processed FS (data MTT-test). According to the degree of sensitivity to irradiation with laser cell lines are as follows: NGOC>R>Ner.

After prolonged action of FCS at a concentration of 5 cells in the darkness survived 96,5-86.2% of PC-12, 103,7-93,0% NGOK and 109,7-87,9% Ner (MTT-test - crystal-violet, respectively). In these conditions, DNA synthesis remained unchanged in cells PC-12 and was reduced in cells Near and NGOC 21.2 and 22.2%, respectively. The observed increase in the number of cells NGOK and Ner under the action of 5 M FS cells in the dark is due most likely to the induction of the Federal Assembly of the proliferative activity of cells. In General, for FS in the absence of irradiation more typical manifestation of the cytotoxic activity than the induction of proliferative.

After laser irradiation, cells treated with FS, there is their death. Found dosevasive cytopathological effect of the drug that allows you to calculate EU50, i.e., to determine the concentration of FS that will kill 50% of cells. These data are shown in table 2. It should be noted that FS, at which the EU50less than 20 M, it is considered effective to suppress tumor growth.

When determining generatesecret after treatment of cells with 5 M FS and laser exposure produces NGOC at low concentrations FS after laser exposure observed stimulation of DNA synthesis, which is greatly reduced in the presence of 5 M BX. The observed increase in DNA synthesis can be explained by the high ability to synthesize DNA and to restore their population surviving at low concentrations FS transformed liver cells and glia.

Thus, FS is vysokotemperaturnym medication for different types of tumor cells. In high concentrations (>5 M) he is a moderate inhibitor of tumor growth and without irradiation. High generatesecret FS can be considered a strong inhibitor of tumor growth under irradiation.

In vivo study of the toxic properties of FS (example 14). LD50on average, taking into account the weighting factor, 210,5322,2 mg/kg, and the dose causing the death of 10% of the test animals (LD50), is 169,87 mg/kg of the Carried out researches allow to classify FS as a "Toxic substance".

In vivo study the biodistribution FS (example 11). With the introduction of FS intraperitoneally to mice with embrocations T36, intertwined into the muscle of the back leg, observed the following patterns in the distribution of connections. After the introduction of the FS into the blood stream, and then redistributed in the organs and tchasov after intraperitoneal administration at a dose of 40 mg/kg and remains for a long time (18-24 hours). Tumor concentration after 18 h after injection is 0.48 M, which is 1.5 times less than the absolute maximum accumulation at high selectivity of accumulation. The ratio of tumor/muscle tissue is 32, and the tumor/skin - 44.

After intravenous administration at a dose of 20 mg/kg to reach the maximum accumulation in the tumor after 0.5 hours (0,32 M), which also remains for a long time (up to 5 hours). The maximum contrast accumulation when administered intravenously is manifested through 3 hours and is for the tumor/muscle tissue 3, and tumor/skin - 4. FS excreted through the day at 98%.

The results of the evaluation of the effectiveness of the drug for PDT of cancer in vivo in mice (example 15) allow us to ascertain the existence of pronounced photodynamic activity "Radachlorin, 0.5% solution for injections" ("Photochlorin") and "Radachlorin, the 0.05% gel".

Drug substance "Liquid extract of chlorins", including sodium salt chlorine (or salt chlorine and other strong inorganic bases), is used to get dosage forms by adding to it various allowed by the State Pharmacopoeia of the Russian Federation additives: calcium carbonate, sucrose gluco, is metilsulfate, gel and water-based foundations and other (examples 4-9).

For external use ointments, liniments, gels, drugs, oil-based, contains permitted by the State Pharmacopoeia of the Russian Federation basis, 5-20% of dimethyl sulfoxide and 0.5-12% substance "Liquid extract of chlorins", or 0.8 to 14% substance "Liquid extract of chlorins" and 86-99,2% dimethyl sulfoxide (examples 8, 9).

The range of values of the content of dimethyl sulfoxide in combination with the basics connected with the fact that when its concentration is less than 5% penetration of the substance into the tissue is small, which reduces the efficiency of PDT. When the concentration of dimethyl sulfoxide more than 20% of the dosage form on other grounds lose their stability during storage. The range of values of the content of a substance is related to the fact that when its concentration is less than 0.5% of the content of the substance in the tissue is not sufficient for effective PDT. When the concentration of the substance more than 12% of the fabric loses its transparency to light radiation, all the light is absorbed in the top layer of tissue that leads to burn at low efficiency of this procedure PDT.

When applied externally, the exposure of the substance on the skin before exposure is 0.5-24 hours. For vblades the drop in the value of the absolute accumulation of the drug due to its redistribution and excretion. In addition, prolonged exposure of the exterior of dosage forms on the skin inconvenient from a clinical point of view.

When intravenous use of the drug is administered as 0.1-1% solution in any allowed by the State Pharmacopoeia of the Russian Federation environments (pyrogen-free water for injection, dimethylsulfoxide, saline etc) drip or bolus. The use of mixtures of substances, diluted more than 0.1%, irrational from the point of view of volumes entered in the body fluid. The use of concentrated solutions of 1% is impossible due to the poor filterability of such solutions at the stage of sterilization through bacterial filters.

To activate the substance FS "Liquid extract of chlorins" use of the semiconductor laser diode module for photodynamic therapy ML-662-SP developed by companies CJSC "MILON" in St. Petersburg) and LLC "SIGMA PLUS", Moscow). This module has the following output [12]:

- capacity: 2.5-3 watts in the fiber 200 µm aperture is 0.22;

- vysokoemkie laser diodes co-production firm "Polaroid" (USA), LLC "SIGMA PLUS" with a maximum wavelength of radiation 6623 nm.

To activate the substance FS can be used m the tion laser pumped by the second harmonic yttrium aluminum garnet YAG:Nd+3with a maximum emission wavelength of 670 nm.

The value supplied energy range from 30 to 3000 joules. When light doses less than 30 j procedure PDT becomes excessively long, because to achieve the optimal effect of scan to be performed on very small areas. When light doses over 3000 j and the most common in clinical practice, the sizes of the tumors observed significant damage to healthy tissue, leading to an increase of the period of regeneration.

The surface density of the supplied energy range from 50 to 2500 j/cm2. When the surface light doses less than 50 j/cm2there is no effect. When the surface light doses above 2500 j/cm2there has been significant damage to healthy tissue, leading to an increase of the period of regeneration.

The wavelength range of the excitation radiation is associated with the technical characteristics of the laser used (6623 nm), the shift of the maximum absorption of the drug depending on the polarity of the environment (654-662 nm) and the content of purpurine 5 in substance (5-20%, the width of the long-wave absorption band at 663-670 nm) (table. 1).

Example 1. Description of physico-chemical properties of f with the smell of seaweed.

To verify properties of FS, "Liquid extract of chlorins", 7,5% thoroughly mixed, a portion of the extract (1 mg) dissolved in 10 ml of ethyl alcohol distillation, 95%, medical or highest purification and measure the optical density at a wavelength of 662 nm (D). Get the value of 0.23. Calculate the molecular extinction (M-1cm-1the formula =D597/(0,004). The resulting value must be in the range 33300-35100. Substituting receive =0,23597/(0,004)= 34328. Consequently, the "Liquid extract of chlorins" contains 7.5% of FS.

The solution FS in ethyl alcohol has a yellow-green color. When passing through the solution layer of the light rays from the lamp medical blue marks MDS 220-75 (TU 16.535.376-79) in the dark place, the solution becomes a ruby-red color.

For the quantitative determination of "Liquid extract of chlorins" thoroughly mixed, a portion of the extract (5 mg) dissolved in 10 ml of ethyl alcohol distillation, 95%, medical or highest purification and measure the optical density at a wavelength of 662 nm (D). Get the value of 2.15. Calculate the content of FS according to the formula: c%=(D59710100)/(342305). The resulting value must be specified. Substituted with,%= (2,1559710100)/(342305)=7,5% (ACC is undertaken hydrochloric acid and the precipitate discarded FS, which is filtered off, dried in vacuum over pjatiokisi phosphorus within 12 h and remove PMR, mass spectra and absorption spectrum in the wavelength range 360-720 nm.

Range PMR FS (Fig. 6): (DMSO-D6, conc. the solution): 9,64, 9,55, 9,52, 9,39, 8,90, 8,79 (with, methochloride e6and purpurine 5), 8,09, 8,04, 7,97, 7,92 (2D, chlorin e6and purpurine 5), at 6.84 (s, meso - purpurine 5), 6,37, 6,32, 6,13, 6,10 (2D ), 5,43 (2s, -meso-), 4,60 (m ), of 4.45 (m, , 3.80, 3,56 (CH, ), 3,75, 3,64, 3,51, 3,46, 3,29, 3,23 (C, nuclear chlorin e6and purpurine 5), 2,38, 2,32 (2 m, 7-), 2,71, 2,20 (2m ), of 1.76 (d, ), 1,72 (t ), 1,63,-1,91 (2C ) M. D.

The mass spectrum of the FS (Fig. 7): e. i. M+(%), 596 (16,0), 566 (9,4), 508 (100,0), 494 (7,3), 447 (9,4), 435 (50,6), 421 (12,8), 405 (6,9), 254 (7,4).

Absorption spectrum of FS in the visible region: () (ethanol), 386 (22310), 406 (113040), 506 (14870), 536 (8925), 608 (7437), 662 (34220).

According to PMR spectrum, the substance contains 80% of chlorin e6, 15% of purpurine 5 and 5% of purpurine 18 (minor signals at 9,25, 9,10, 8,71, 7,84, 3,55, 3,32, 3,04 memorial plaques), which corresponds to a patentable composition. According to the mass spectrum contains peaks of molecular ions 596 from chlorin e6and 566 from purpurine 5. In the absorption spectrum has a band at 662 nm with the magnitude of the absorption, is well correlated with the molecular extinction pattern FS (34230).

Therefore, studied the act of chlorins", 6,5%.

Process the biomass of Spirulina (2 kg) acetone (3x2 l) to complete the extraction of chlorophyll a, filtered biomass, process, extract hydrochloric acid (30 ml) to remove chlorophyll molecules of the magnesium ion, neutralize the extract is precipitated and filtered pheophytin a (8 g), then hydrolyzing pheophytin and in a mixture of hydrochloric acid-acetone-hexane, which pheophytin dissolved in a mixture of 50 ml of acetone, 5 ml of hexane and 40 ml of hydrochloric acid (37%), heat the mixture to a temperature of 40oC and stirred for 1 hour, then add hexane (50 ml) and the organic phase is washed with a mixture of acetone and concentrated hydrochloric acid 2:1 (h ml), the aqueous phase is washed with hexane (5x40 ml), then neutralized aqueous phase containing pheophorbide and an excess of an aqueous solution of sodium citrate (three-, two - or one-deputizing), separating the rolled deformed but by filtration, washed with water (I ml), periostat it from a mixture of acetone-water, air-dried until constant weight (yield pheophorbide and to 4.2 g, 7.1 mm, 77%), then dissolve pheophorbide and (2.7 g, 4,56 mm) in acetone (100 ml), add a strong inorganic base in aqueous solution with a concentration of 0.05%, 25 ml), stirred at 60oC for 5 min, add the l is oC for 90 min, neutralized mixture of diluted hydrochloric acid (2%, 250 ml), separating the precipitate of chlorin e6by centrifugation, washed it with distilled water (5 x 10 ml) until the disappearance of the acid reaction, gain of 1.85 g (2,96 mm, 65%) of chlorin e6, periostat chlorin e6from acetone to separate linear tetrapyrroles, filtered chlorin e6and washed 3 times with distilled water, heated chlorin e6in sealed containers at 40oWith within 30 days, cooled and added 1% sodium hydroxide solution to pH 7.5, receiving FS with the 15% of purpurine 5, 80% of chlorin e6and 5% of purpurine 18 (chlorin p6), which Ousterhout distilled water to 6.5% of the content of FS, receiving 14.2 g (50%) FS in the form of 6.5% "Liquid extract of chlorins".

Range PMR received "Liquid extract of chlorins" (Fig. 6): (DMSO-D6, conc. the solution): 9,64, 9,55, 9,52, 9,39, 8,90, 8,79 (s, meso-chlorin e6and purpurine 5), 8,09, 8,04, 7,97, 7,92 (2D, chlorin e6and purpurine 5), at 6.84 (s, meso purpurine 5), 6,37, 6,32, 6,13, 6,10 (2D ), 5,43 (2s ), 4,60 (m ), of 4.45 (m, ), 3,80, 3,56 (CH, ), 3,75, 3,64, 3,51, 3,46, 3,29, 3,23 (C, nuclear chlorin e6and purpurine 5), 2,38, 2,32 (2m ), 2,71, 2,20 (2m, 7-), 1,76 (d ), 1,72 (t ), 1,63, -1,91 M. D.

In substance,32, 3.04 from M. D.).

The mass spectrum of the obtained substance (Fig. 7): e.i., M+(%), 596 (16,0), 566 (9,4), 508 (100,0), 494 (7,3), 447 (9,4), 435 (50,6), 421 (12,8), 405 (6,9), 254 (7,4).

Absorption spectrum in the visible region (Fig. 8): () (ethanol), 386 (22320), 406 (113110), 506 (14880), 536 (8930), 608 (7440), 662 (34230).

Example 3. Getting FS in the form of substance "Liquid extract of chlorins", 7,5%.

Process the biomass of Spirulina (2 kg) acetone (3x2 l) to complete the extraction of chlorophyll a, separate the biomass by centrifugation, treated with the extract hydrochloric acid (30 ml) to remove chlorophyll molecules of the magnesium ion, neutralize the extract is precipitated and filtered pheophytin a (8 g), then hydrolyzing pheophytin and in a mixture of hydrochloric acid-acetone-hexane, which pheophytin dissolved in a mixture of 100 ml of acetone, 50 ml of hexane and 80 ml of hydrochloric acid (37%), heat the mixture to a temperature of 60oC and stirred for 20 min, then was added hexane (100 ml) and the organic phase is washed with a mixture of acetone and concentrated hydrochloric acid 5:1 (g ml), the aqueous phase is washed with hexane (5x40 ml), then neutralized aqueous phase containing pheophorbide and an excess of an aqueous solution of sodium citrate (three-, two - or one-deputizing), separating the rolled deformed but by filtration, washed with s is%), next, dissolve pheophorbide and (2.7 g, 4,56 mm) in acetone (100 ml), add a strong inorganic base in aqueous solution with a concentration (1%, 25 ml), stirred at 30oC for 30 min, add an additional amount of a strong inorganic bases (20%, 25 ml) in aqueous solution, heated at 60oC for 20 min, neutralized mixture of diluted hydrochloric acid (2%, 250 ml), separating the precipitate of chlorin e6by centrifugation, washed it with distilled water (5 x 10 ml) until the disappearance of the acid reaction, get 1,67 g (2,67 mm, 55%) of chlorin e6, periostat chlorin e6from acetone to separate linear tetrapyrroles, filtered chlorin e6and washed 3 times with distilled water, heated chlorin e6in a sealed vessel at 100oC for 1 hour, cooled and added 1% solution of potassium hydroxide to a pH of 8.5, resulting in receive FS with 2% of purpurine 5, 82% of chlorin e6and 16% of purpurine 18 (chlorin p6), Ousterhout distilled water up to 7.5% of the content of FS, receiving 11.1 g (50%) FS in the form of 7.5% paste.

The spectra were obtained substance similar spectra, sample 2, and represent superpos is With - obtain the "Liquid extract of chlorins", 7,5%.

FS 2% of purpurine 5, 82% of chlorin e6and 16% of purpurine 18 (chlorin p6in the form of 7.5% paste from the previous example is subjected to gel filtration on a column of Sephadex G10 diameter 50 mm and height 100 mm, elwira 1% potassium hydroxide solution until the content of chlorin e6- 90%, purpurine 5 - 5% and purpurine 18 - 5%. Add a dilute solution of hydrochloric acid and the precipitate discarded FS, Ousterhout pyrogen-free water for injection to the content of the photosensitizer to 7.5 wt.% and obtain 6.8 g "Liquid extract of chlorins", to 7.5%. Electronic product spectrum, see Fig. 8.

Example 5. A special case of receiving FS - receiving dosage forms "Radachlorin, 0.1% solution for injections".

After gel filtration the solution FS from example 4 was added a dilute solution of hydrochloric acid and the precipitate discarded FS, the residue is filtered off, add concentrated sodium hydroxide solution in pyrogen-free water for injection to pH 7.5 and pyrogen-free water for injection to the content of FS 0,1%, after which the solution is filtered off from bacteria a bacterial microporous filter "Millipore" with the pores diameter is 0.22 m Solution - 500 ml. E range of p is Rin, 0.5% solution for injections" ("Potohari").

After gel filtration the solution FS from example 4 was added a dilute solution of hydrochloric acid and the precipitate discarded FS, the residue is filtered off, add a concentrated solution of potassium hydroxide to pH 7, then when the control by pH-meter lead to the solution of N-methyl-D-glucamine to pH 8.5, was added pyrogen-free water for injection to the content of the photosensitizer 0.5 wt.% and filtered off from bacteria a bacterial microporous filter "Millipore" with the pores diameter is 0.22 m Solution - 100 ml. Electronic product spectrum, see Fig. 8.

Example 7. A special case of receiving FS - receiving dosage forms "Radachlorin, 1% solution for injections".

After gel filtration the solution FS from example 4 was added a dilute solution of hydrochloric acid and the precipitate discarded FS, the residue is filtered off, add a concentrated solution of sodium hydroxide to pH 8.5, and then add pyrogen-free water for injection to the content of the photosensitizer 1 wt. % and filtered off from bacteria a bacterial microporous filter "Millipore" with a pore diameter of 0,22 m Output - 50 ml. Electronic spectrum of the product is.

After gel filtration the solution FS from example 4 was added a dilute solution of hydrochloric acid and the precipitate discarded FS, this precipitate was separated by centrifugation, Ousterhout pyrogen-free water for injection to the content of the photosensitizer to 6.5 wt.%, and then proceed according to the options:

Option (a). To 75 ml of water and 5 g of dimethylsulfoxide was added at room temperature, 0.3 g Pemulen TR1 or Carbopol 2020 (BF Goodrich, UK) and stirred for1/4-8 hours. Add an aqueous solution of alkali to a pH of 5. Resuspending gel with the addition of "Liquid extract of chlorins", 6.5% and water so that the content of chlorin e6in the finished gel was 0.05%, and vaccum 5 min at 10-50 mm RT. senior Exit - 100 g of gel.

Option (b). To 70 ml of water are added at room temperature, 5 g of dimethylsulfoxide and "Liquid extract of chlorins", 6.5 percent, so that the contents of chlorin e6in the finished gel was 0.05%, and then add 15 g Aculyn 33A (ISP, USA). Mix until smooth and add an aqueous solution of alkali to a pH of 5. Vaccum 5 min at 10-50 mm RT. senior Exit - 100 g of gel.

After gel filtration the solution FS from example 4 was added a dilute solution of hydrochloric acid and the precipitate discarded FS, the residue division is further possible options:

Option (b). To 60 ml of water and 20 g of dimethylsulfoxide was added at room temperature, 0.7 g Pemulen TR1 or Carbopol 2020 (BF Goodrich, UK) and stirred for1/4-8 hours. Add an aqueous solution of triethanolamine to a pH of 8.5. Resuspending gel with the addition of "Liquid extract of chlorins", 7.5% and water so that the content of chlorin e6in the finished gel was 1%, and vaccum 5 min at 10-50 mm RT. senior Exit - 100 g of gel.

Option (d). To 55 ml of water are added at room temperature, 20 g of dimethyl sulfoxide and "Liquid extract of chlorins", 7.5%, and so that the contents of chlorin e6in the finished gel was 1%, and then add 15 g Aculyn 33A (ISP, USA). Mix until smooth and add an aqueous solution of triethanolamine to a pH of 8.5. Vaccum 5 min at 10-50 mm RT. senior Exit - 100 g of gel.

Example 9. A special case of receiving FS - receiving dosage forms "Radachlorin, solution in dimethyl sulfoxide for external use".

Option (a). After gel filtration in example 4, to the mixture was added diluted hydrochloric acid and the precipitate discarded FS, the residue is filtered off, Ousterhout pyrogen-free water for injection to the content of FS 7.5 wt.%, and 14 g of the obtained Liquid extract hariom solution was 1%, and mix until smooth. Output - 100 g of solution.

Option (b). After gel filtration in example 4, to the mixture was added diluted hydrochloric acid and the precipitate discarded FS, the residue is filtered off, Ousterhout pyrogen-free water for injection to the content of FS 7.5 wt.% and 0.8 g of this "Liquid extract of chlorins" at room temperature add to 99.2 g of dimethyl sulfoxide to the content of chlorin e6in the finished solution was 0.05%, and mix until smooth. Output - 100 g of solution.

Example 10. To identify purpurine 5 the reaction mass of example 2 is subjected to gel filtration on a column of Sephadex G10, elwira 1% solution of N-methyl-D-glucamine. Get 3 factions, of which the first and second contain purpurine 5. They neutralize, precipitate is filtered off, dissolved in chloroform-methanol 1:1 and etherification with diazomethane. Washed with water, the organic phase is separated, dried with anhydrous magnesium sulfate, concentrated by evaporation in a vacuum and chromatographic on silica gel Merck, Kieselgel, 0,04-0,063, collecting the last (least mobile) faction. The obtained dimethyl ether of purpurine 5 (10.1% in terms of dry reaction mass is taken to esterification) when neobhodimo, ), PC 6.82 (s, - meso), 6,34, 6,31, 6,19, 6,16 (2D ), of 4.54 (m, ), 4,46 (m ), 3,61 (, ), 4,20, 3,81, 3,57, 3,53, 3,47 (5C, and nuclear ), 2,38, 2,35 (2m ), 2,68, 1,85, (2m ), 1,73 (d ), 1,70 (t ) M. D.

Mass spectrum (Fig. 16): e.i., M+(%), 594 (8,6), 566 (100,0), 505 (5,1), 491 (9,8), 475 (8,2), 463 (1,7), 447 (1,4), 433 (1,7), 403 (2,0), 262 (5,0).

Absorption spectrum in the visible region (Fig. 17): () (chloroform), 408 (117200), 501 (11380), 542 (9830), 617 (6720), 668 (35200).

Dimethyl ether of purpurine 5 is dissolved in acetone and added conc. hydrochloric acid (37%) in the ratio 1:2. Stirred for 2 hours at 25oWith, neutralize, purpurine 5 filtered off, washed with water, dissolved in 10% solution of N-methyl-D-glucamine and subjected to gel filtration on a column of Sephadex G10, elwira 1% solution of N-methyl-D-glucamine, collecting the second fraction, neutralize, precipitate is filtered off, washed with water, dried over pjatiokisi phosphorus to constant weight. Get purpurine 5 (5.2% in terms of dry reaction mass is taken to esterification).

PMR-spectrum (Fig. 12): (DMSO-D6, conc. solution): of 9.55, 9,39, 8,79 (, ), 8,09, 8,04, 7,97, 7,92 (2D ), 6,84 (, ), 6,37, 6,32, 6,13, 6,10 (2D ), 4,60 (m ), of 4.45 (m, ), 3,55 (K ), 3,75, 3.46 in, 3,23 (nuclear ), 2,38, 2,32 (2m ), 2,71, 2,20 (2m ), of 1.76 (d, ), 1,72 (t, 4-) M. D.

Mass spectrum (Fig. 13): e.i., M+(%), 566 (8,2), 494 (100,0), 447 (9,1), 435 (49,6), 421 (12,7), 405 (6,6), 254 (7,1).

The mice Bulb/c intraperitoneally injected 769,2 mg/kg 6,5% substance "Liquid extract of chlorins" from example 2 (50 mg/kg, calculated on the anhydrous substance chlorine). After 3 hours after injection produce the slaughter of mice (each group 3 mice). The material of the liver, kidney, spleen, lung, small intestine, tumor, surrounding muscle tissue as well as blood, urine, feces from the colon, by weight, 100 mg are subjected to a thorough homogenization in a glass homogenizer with the addition of 4 ml of physiological solution. For the study of biological liquids (blood, urine) take 0.1 ml each, followed by dissolving in 4 ml of saline. The resulting homogenates research on spectrofluorimeter firm Perkin-Elmer (model MPF-44A).

Similarly conduct a study of dosage forms "Radachlorin, 0.5% solution for injections" ("Potohari"), in homogenates of organs and tissues of mice, to which the drug is administered intraperitoneally at a dose of 50 mg/kg, with the slaughter of animals 3 h after injection.

In both cases there is a shift mo compared with 0.01 M borate buffer solution, the pH of 9.2 and 5-6 nm compared with 0.01 M borate buffer solution, pH of 9.2 with the addition of 1% serum albumin human), indicating that metabolism "Radachlorin, 0.5% solution for injections" ("Photochlorin") (Fig. 5).

This phenomenon in the spectra of fluorescence is different than the common broadening of the spectrum and the shift in the long wavelength region due to the influence of the hydrophobicity of the environment (for example, after hydrophobic interaction with proteins, lipoproteins). The shift of the maximum intensity is observed without ening strip or with a small broadening, which is typical for the formation of a new connection. The spectra of the fluorescence of purpurine 5 in 0.01 M borate buffer solution, pH of 9.2 with the addition of 1% serum albumin human is characterized by the presence of bands at 670 nm.

In the blood, lung parenchyma, as well as in skin and tumors observed broadening of the spectra at 1.4-1.5 times the wavelength 669 nm, indicating the presence in the homogenates of the mixture "Radachlorin, 0.5% solution for injections" ("Photochlorin") (complex with proteins and metabolites.

Adding "Radachlorin, 0.5% solution for injections" ("Photochlorin") directly in tubes with tissue homogenates intact the Radachlorin, 0.5% solution for injections" ("Photochlorin") (offset in long-wavelength region without broadening of the spectrum), and only in the skin homogenate there has been a slight increase in the half width of the spectrum at 1.15 times, which indicates the presence in the sample mixture Potohari" - metabolite.

By increasing the concentration of the "Radachlorin, 0.5% solution for injections" ("Photochlorin") up to 5-10 M in homogenates of organs, almost all samples recorded the presence of a mixture of chlorin e6- purpurine 5 (displacement spectra in the wavelength region by increasing the half-width of the spectra 1.15-1,05 times).

Thus, we can assume that the formation of the metabolite adding "Radachlorin, 0.5% solution for injections" ("Photochlorin") in the homogenates depends on the concentration of the drug and the enzyme activity homogenized tissue.

These experiments demonstrate the transformation of chlorin e6in purpurine 5 in vivo and ex vivo. This transformation similar to the transformation of chlorin e6in purpurine 5 when heated.

Example 12. The distribution coefficient n-octanol/phosphate buffer, pH 7.4. Shake for 20 with 300 ml of n-octanol and 300 ml of phosphate buffer solution, pH 7.4 and doctorale (8 ml) dissolving an aliquot of the Federal Assembly of a volume of 0.1 ml with a concentration of FS 5 mg/ml and determine the absorption maximum at a wavelength of 406 nm. Get the values of Dtooand Dtobwhere o - n-octanol, b - phosphate buffer to control. Equilibrium distributions of the n-octanol/phosphate buffer to achieve, shaking at 20oWith 2 ml of phosphate buffer and 8 ml of n-octanol with 0.1 ml of FCS for 20 sec, followed by centrifugation for 10 min at 10000 rpm by Measuring the optical density of each phase at 406 nm, gain values Daboutand Dbwhere o - n-octanol, b - phosphate buffer.

TOpdetermined by the formula:

Kp=(DoVoDtooVtoo)/(DbVbDtobVtob), where Vabout- the volume of octanol taken to determine the equilibrium distribution (8 ml), Vaboutto- the volume of octanol saturated with water, taken to control for absorbance of aliquots (8 ml), Vb- the volume of the buffer is taken to determine the equilibrium distribution (2 ml), Vtob- volume of buffer saturated with octanol taken to control for absorbance of aliquots (2 ml). The experiment is carried out 3 times and the obtained values TOpaverage.

Get the value 1,40,3.

Example 13. Definition of photocytotoxicity (biological actively (Potohari").

In use the kelp firm "Flow Lab (UK), CO2incubator Flow Lab (UK), MultiScan Bio-Tek Instruments" (USA), environment and serum company "Panico" (Russia).

One experience of one cell line passedout in two 48-hole tablet: for the irradiation of the laser and for "dark" experience. The next day, cells are able confluent add the drug and tablets next thermostatic in black paper. Explore drug concentrations of 0.1, 0.5, and 2.0 and 5.0 M. in 3 hours after addition of the drug irradiated cells with a laser, and the exposure dose of 50 j/cm2and after 39 hours then spend the MTT-test and incubation with14With-thymidine to measure the magnitude of DNA synthesis (and in the "dark" tablet too). In all cases the upper part of the tablet is used for the MTT test, and the bottom for the measurement of DNA synthesis, as well as the number of cells after staining the crystal-Violeta.

Are given in table. 2 the results are average of 4 parallel experiments.

Example 14. The study of the toxic properties of the substance "Liquid extract of chlorins" and dosage forms "Radachlorin, 0.5% solution for injections" ("Potohari") in vivo.

Toxicity study by intravenous introduction is the shaft vivarium conditions and feeding is carried out in accordance with the Order of the USSR Ministry of 1179 from 10.10.83 of the year "On approval of regulations of the cost of feed for laboratory animals in health care facilities".

Toxicity is determined by the deaths of animals, after calculating sredneseriynoe dose - LD50. The calculation carried out by statistical methods, recommended by the State Pharmacopoeia XI edition (1,3). On the basis of LD50determine the identity of the analyte to a specific class of toxicity on the Hodge and Sterner. They also hold into account the reactions of intoxication during the experiment.

In the experience of selected mice of 12 individuals (6 males and 6 females) for each tested dose FS. To determine LD50FS examine the following doses: 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 mg/kg of solution with a concentration of 5 mg/ml FS administered to mice intravenously, by varying the dose-volume filesystem.

Get the value LD50equal 210,5322,2 mg/kg LD10equal 169,87 mg/kg

Example 15. Biological activity in vivo using dosage forms "Radachlorin, 0.5% solution for injections" ("Potohari").

The study of the photodynamic activity of the dosage form "Radachlorin, 0.5% solution for injections" ("Photochlorin") carried out on mice of Balb/c with interwoven into the muscle of the hind legs by embrocations T36. Mice weight - 20-21, radiation treatment carried out by the diode laser ML-662 - radiation.

The drug is administered intraperitoneally at a dose of 40 mg/kg of body weight, which corresponds to a sufficient therapeutic dose. For the procedure of irradiation of mice injected in the ether anaesthesia. The weight of tumors in the control and experimental group at the time of the experiment varies from 0.9 to 1, the Irradiation is carried out through 5-6 hours after administration of the Federal Assembly. Every animal, except the control, exposed to a single radiation treatment, then observations are within one month after the exposure, the recorded area of the tumor necrosis and General physiological condition.

The average density of the exposure dose is 150 or 300 j/cm2.

The best results in the form of complete tumor necrosis, crust formation after 1 week after PDT and its drop in 1.5 months after PDT see in the group that received a light dose of 300 j/cm2.

Example 16. Treatment of basal cell skin cancer using dosage forms "Radachlorin, 0.5% solution for injections" ("Potohari").

Cytological examination of scraping establish the diagnosis: basal cell skin cancer. The drug is administered intravenously at the rate of 0.7 mg/kg weight of the patient Poluchenie tumor diode laser ML-662-SP with a wavelength of 662 nm with a surface dose of 50 j/cm2. During injection and laser irradiation is not noted any adverse reactions. 2 hours after irradiation on the tumor location see hearth dark brown color with an area of redness around 1-2 refer To the end of the first day on the site of the tumor is formed necrosis in the form of a dry brown dark brown (scab). After 2-3 weeks there is a rejection of the scab and 2 weeks after exclusion of scab at the site of the former basal cell carcinoma is complete epithelialization of the skin defect at the site of the tumor with good cosmetic effect.

Example 17. Treatment of basal cell skin cancer using dosage forms "Radachlorin, 0.05% gel".

Cytological examination of scraping establish the diagnosis: basal cell skin cancer. Gel thin layer of the tumor as possible without hurting the healthy part of the skin. Irradiation is carried out through 20-40 minutes after applying the gel. Procedure carry out irradiation with diode laser ML-662-SP (production Milon-Sigma Plus", Russia) with a wavelength of 662 nm. The density of the exposure dose is 2500 j/cm2. 2 hours after irradiation on the tumor location see hearth dark brown with the dark brown color (scab). After 2 weeks there is a rejection of the scab and 2 weeks after exclusion of scab at the site of the former basal cell carcinoma is complete epithelialization of the skin defect at the site of the tumor with good cosmetic effect.

Example 18. Tattoo removal using dosage forms "Radachlorin, 0.5% solution in dimethyl sulfoxide for external use". Put the solution on the cloth and put it over the tattoo, cover with black paper or thin aluminum foil and fixed for 30 minutes to Remove excess solution from the surface with a cotton swab moistened with alcohol. Procedure carry out irradiation with diode laser ML-662-SP (production Milon-Sigma Plus", Russia) with a wavelength of 662 nm, and exposure to lead, repeating a line drawing, taking care not to touch the surrounding line of fabric. The density of the exposure dose is 120 j/cm2. Within 1 hour after irradiation on the picture see the redness and swelling. By the end of 2 days formed a "picture" of a dark brown color with an area of redness around to 1 mm After 2 weeks on the picture is formed necrosis in the form of a dry crust is dark brown. After 2 weeks is peeling of the crust together with reboots repeated sessions. In the PDT on the spot tattoo in 6 weeks is formed of a soft pink fabric, little different from the surrounding skin, with good cosmetic effect.

1. Photosensitizer for photodynamic therapy, characterized in that it includes an alkaline salt 13-carboxy-17-[2-carboxyethyl] -15-carboxymethyl-17,18-TRANS-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylpiperidine (chlorin e6) formula

< / BR>
in the amount of 80-90%; alkaline salt 13-carboxy-17-[2-carboxyethyl] -15-formyl-17,18-TRANS-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylpiperidine (purpurine 5) formula

< / BR>
in the amount of 5-20%; and alkaline salt 13-carboxy-17-[2-carboxyethyl] -15-carboxy-17,18-TRANS-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylpentane (chlorin p6) formula

< / BR>
in the number - rest.

2. The photosensitizer under item 1 in the form of an aqueous solution or gel.

3. The photosensitizer on the PP. 1 and 2, characterized in that the alkali metal used sodium.

4. The photosensitizer on the PP. 1 and 2, characterized in that the alkali metal used potassium.

5. A method of obtaining a photosensitizer on PP. 1-4, according to which process the biomass of Spirulina ACE the act acid prior to the removal of the chlorophyll molecules of the magnesium ion and hydrolysis failboy ester group, the extract is neutralized buffer solution, filtered fallen pheophytin a and hydrolyzing it with a mixture of hydrochloric acid-acetone-hexane, and 1 g of the crude pheophytin take a 6-16 ml of acetone, 0.6 to 6 ml of hexane and 5-10 ml of concentrated hydrochloric acid, heat the mixture to a temperature of 40-60oC, stirred for 20 min - 1 h, add hexane 6-16 ml) and the organic phase is washed with a mixture of acetone and concentrated hydrochloric acid (2-10): 1, the aqueous phase is washed with hexane, neutralize the combined aqueous phase containing pheophorbide a, the excess aqueous buffer solution, separating the rolled pheophorbide a by filtration, washed with water, periostat from a mixture of acetone-water, air-dried until constant weight, dissolve pheophorbide a in acetone, add an aqueous solution of a strong inorganic base with a concentration of 0.05 to 1.00%, and then stirred at 30 to 60oC for 5-30 min, add an additional amount of an aqueous solution of a strong inorganic base with a concentration of 1-50%, heated at 40-60oFor 20-90 min, neutralized mixture of diluted hydrochloric acid, the precipitate is separated by centrifugation, washed with distilled water until the disappearance of the acid reaction, Peresada heated in sealed containers within the temperature range 40-100oC for 1 h for 30 days and cooled, followed, if necessary, by adding water and grounds.

6. A method of obtaining a photosensitizer under item 5, wherein after cooling and adding water and the base of the photosensitizer is subjected to gel filtration to the content of chlorin e680-90%, purpurine 5 5-20% and chlorin p6- the rest, add a dilute solution of hydrochloric acid and the precipitate discarded photosensitizer, filters it out, washed with water, Ousterhout solution of a base to a pH of 7.5-8.5 and pyrogen-free water until the content of the photosensitizer of 6.5-7.5 wt. %.

7. A method of obtaining a photosensitizer on PP. 5 and 6, characterized in that the photosensitizer is added pharmaceutically acceptable additives, solution Ousterhout and filtered from bacteria.

8. A method of obtaining a photosensitizer on PP. 5 and 6, characterized in that the photosensitizer is dispersed in a gel base, add pharmaceutically acceptable additives and water and Ousterhout.

9. A method of obtaining a photosensitizer on PP. 5 and 6, characterized in that the photosensitizer is mixed with dimethylsulfoxide.

 

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FIELD: medicine.

SUBSTANCE: method involves introducing 0.1-0.3 ml of photosensitizing gel preliminarily activated with laser radiation, after having removed neovascular membrane. The photosensitizing gel is based on a viscoelastic of hyaluronic acid containing khlorin, selected from group containing photolon, radachlorine or photoditazine in the amount of 0.1-2% by mass. The photosensitizing gel is in vitro activated with laser radiation having wavelength of 661-666 nm during 3-10 min with total radiation dose being equal to 100-600 J/cm2. The gel is introduced immediately after being activated. To compress the retina, vitreous cavity is filled with perfluororganic compound or air to be further substituted with silicon oil. The operation is ended with placing sutures on sclerotomy and conjunctiva areas. Compounds like chealon, viscoate or hyatulon are used as viscoelastic based on hyaluronic acid. Perfluormetylcyclohexylperidin, perfluortributylamine or perfluorpolyester or like are used as the perfluororganic compound for filling vitreous cavity.

EFFECT: excluded recurrences of surgically removed neovascular membrane and development of proliferative retinopathy and retina detachment; retained vision function.

3 cl, 5 dwg

FIELD: medicine.

SUBSTANCE: method involves making incision in conjunctiva and Tenon's capsule of 3-4 mm in size in choroid hemangioma projection to sclera 3-4 mm far from limb. Tunnel is built between sclera and Tenon's capsule to extrasclerally introduce flexible polymer magnetolaser implant through the tunnel to the place, the choroid hemangioma is localized, after performing transscleral diaphanoscopic adjustment of choroid hemangioma localization and size, under visual control using guidance beam. The implant has permanent ring-shaped magnet in the center of which a short focus scattering lens of laser radiator is fixed. The lens is connected to light guide in soft flexible envelope. The permanent implant magnet is axially magnetized and produces permanent magnetic field of 2-3 mTesla units intensity. It is arranged with its north pole turned towards the choroid hemangioma so that extrascleral implant laser radiator disposition. The other end of the implant is sutured to sclera 5-6 mm far from the limb with two interrupted sutures through prefabricated openings. The implant is covered with conjunctiva and relaxation sutures are placed over it. Light guide outlet is attached to temple using any known method. 0.1-1% khlorin solution is injected in intravenous bolus dose of 0.8-1.1 mg/kg as photosensitizer and visual control of choroid hemangioma cells fluorescence and fluorescent diagnosis methods are applied. After saturating choroid hemangioma with the photosensitizer to maximum level, transscleral choroid hemangioma laser radiation treatment is carried out via laser light guide and implant lens using divergent laser radiation at wavelength of 661-666 nm with total radiation dose being equal to 30-120 J/cm2. The flexible polymer magnetolaser implant is removed and sutures are placed on conjunctiva. Permanent magnet of the flexible polymer magnetolaser implant is manufactured from samarium-cobalt, samarium-iron-nitrogen or neodymium-iron-boron system material. The photosensitizer is repeatedly intravenously introduced at the same dose in 2-3 days after the first laser radiation treatment. Visual intraocular neoplasm cells fluorescence control is carried out using fluorescent diagnosis techniques. Maximum level of saturation with the photosensitizer being achieved in the intraocular neoplasm, repeated laser irradiation of the choroid hemangioma is carried out with radiation dose of 30-60 J/cm2.

EFFECT: enhanced effectiveness of treatment.

4 cl

FIELD: medicine.

SUBSTANCE: method involves creating tunnel between sclera and Tenon's capsule in intraocular neoplasm projection. Intraocular neoplasm localization and size is adjusted by applying transscleral diaphanoscopic examination method. 0.1-0.3 ml of photosensitizing gel based on viscoelastic of hyaluronic acid, selected from group containing chealon, viscoate or hyatulon, is transsclerally introduced into intraocular neoplasm structure using special purpose needle in dosed manner. The photosensitizing gel contains khlorin, selected from group containing photolon, radachlorine or photoditazine in the amount of 0.1-1% by mass. Flexible polymer magnetolaser implant is extrasclerally introduced into the built tunnel in intraocular neoplasm projection zone under visual control using guidance beam. The implant has permanent ring-shaped magnet axially magnetized and producing permanent magnetic field of 3-4 mTesla units intensity, in the center of which a short focus scattering lens of laser radiator is fixed. The lens is connected to light guide in soft flexible envelope. The implant is arranged with its north pole turned towards the intraocular neoplasm so that implant laser radiator lens is extrasclerally arranged in intraocular neoplasm projection zone. The implant light guide is sutured to sclera 5-6 mm far from the limb with single interrupted suture. The implant is covered with conjunctiva and relaxation sutures are placed over it. Light guide outlet is attached to temple using any known method. Visual control of intraocular neoplasm cells is carried out by applying fluorescence and fluorescent diagnosis methods. After saturating the intraocular neoplasm with the photosensitizer to maximum saturation level, transscleral intraocular neoplasm laser radiation treatment is carried out via laser light guide and implant lens using divergent laser radiation at wavelength of 661-666 nm. The treatment course being over, the flexible polymer magnetolaser implant is removed and sutures are placed on conjunctiva. Permanent magnet of the flexible polymer magnetolaser implant is manufactured from samarium-cobalt, neodymium-iron-boron or samarium-iron-nitrogen. 0.1-1% khlorin solution as photosensitizer, selected from group containing photolon, radachlorine or photoditazine, is additionally intravenously introduced in 2-3 days at a dose of 0.8-1.1 mg/kg and repeated laser irradiation of the intraocular neoplasm is carried out with radiation dose of 30-45 J/cm2 15-20 min later during 30-90 s.

EFFECT: complete destruction of neoplasm; excluded its further growth.

4 cl

FIELD: medicine.

SUBSTANCE: method involves applying transscleral diaphanoscopic examination method for adjusting intraocular neoplasm localization and size. Rectangular scleral pocket is built 2/3 times as large as sclera thickness which base is turned from the limb. Several electrodes manufactured from a metal of platinum group are introduced into intraocular neoplasm structure via the built scleral pocket. Next to it, intraocular neoplasm electrochemical destruction is carried out in changing electrodes polarity with current intensity of 100 mA during 1-10 min, and the electrodes are removed. Superficial scleral flap is returned to its place and fixed with interrupted sutures. 0.1-2% aqueous solution of khlorin as photosensitizer, selected from group containing photolon, radachlorine or photoditazine, is intravenously introduced at a dose of 0.8-1.1 mg/kg. Visual control of intraocular neoplasm cells is carried out by applying fluorescence and fluorescent diagnosis methods. After saturating the intraocular neoplasm with the photosensitizer to maximum saturation level, transpupillary laser radiation of 661-666 nm large wavelength is applied at a dose of 30-120 J/cm2. the operation is ended with placing sutures on conjunctiva. Platinum, iridium or rhodium are used as the metals of platinum group. The number of electrodes is equal to 4-8. 0.1-1% khlorin solution, selected from group containing photolon, radachlorine or photoditazine, is additionally repeatedly intravenously introduced in 2-3 days at a dose of 0.8-1.1 mg/kg. Visual control of intraocular neoplasm cells is carried out by applying fluorescence and fluorescent diagnosis methods. After saturating the intraocular neoplasm with the photosensitizer to maximum saturation level, repeated laser irradiation of the intraocular neoplasm is carried out with radiation dose of 30-45 J/cm2.

EFFECT: complete destruction of neoplasm; excluded tumor recurrence; reduced risk of tumor cells dissemination.

3 cl, 3 dwg

FIELD: medicine.

SUBSTANCE: method involves intravenously administering 0.1-1% aqueous solution of khlorin, selected from group containing photolon, radachlorine or photoditazine at a dose of 0.2-0.5 mg/kg or 0.2-1% aqueous solution of porphyrin like photogem at a dose of 0.2-1 mg/kg. Laser irradiation of blood is carried out 5-15 min later after beginning photosensitizer injection into cubital vein of one arm via laser light guide set in advance in the cubital vein of the other arm during 10-40 min at wavelength of 661-666 nm and power of 20-50 mW one session per day during 3-10 days with the aqueous solution of khlorin used as the photosensitizer, or laser irradiation of blood with wavelength equal to 630-633 nm during 10-45 min with power of 20-50 mW one session per day with the aqueous solution of porphyrin used as the photosensitizer. Repeated intravenous administration of photosensitizer is carried out 1-3 months later combined with repeated laser irradiation of blood.

EFFECT: reduced risk of tumor cells dissemination and metastasis development.

2 cl

FIELD: medicine.

SUBSTANCE: method involves intravitreously introducing two electrodes into intraocular neoplasm after carrying out vitrectomy and retinotomy to expose the intraocular neoplasm. The electrodes are manufactured from platinum group metal. Electrochemical destruction is carried out with current intensity of 100 mA during 1-10 min or 10 mA during 10 min in changing electrodes polarity and their position in the intraocular neoplasm space, and the electrodes are removed. 0.1-1% aqueous solution of khlorin as photosensitizer, selected from group containing photolon, radachlorine or photoditazine, is intravenously introduced at a dose of 0.8-1.1 mg/kg. Visual control of intraocular neoplasm cells fluorescence is carried out by applying fluorescent diagnosis methods. After saturating the intraocular neoplasm with the photosensitizer to maximum saturation level, intravitreous laser radiation is carried out in parallel light beam of wavelength equal to 661-666 nm is applied at a dose of 30-120 J/cm2.The transformed retina and tumor destruction products are intravitreally removed. Boundary-making endolasercoagulation of retinotomy area is carried out after having smoothed and compressed retina with perfluororganic compound. The operation is finished with placing sutures on sclerotomy and conjunctiva. Platinum, iridium or rhodium are used as the platinum group metals. Another embodiment of the invention involves adjusting position and size of the intraocular neoplasm in trans-scleral diaphanoscopic way. Rectangular scleral pocket is built above the intraocular neoplasm to 2/3 of sclera thickness with its base turned away from limb. Several electrodes are introduced into intraocular neoplasm structure via the built bed. The electrodes are manufactured from platinum group metal. Electrochemical destruction is carried out with the same current intensity in changing electrodes polarity and their position in the intraocular neoplasm space, and the electrodes are removed. Superficial scleral flat is returned to its place and fixed with interrupted sutures. 0.1-1% aqueous solution of khlorin as photosensitizer, selected from group containing photolon, radachlorine or photoditazine, is intravenously introduced at a dose of 0.8-1.1 mg/kg after having carried out vitrectomy and retinotomy. Visual control of intraocular neoplasm cells fluorescence is carried out by applying fluorescent diagnosis methods. After saturating the intraocular neoplasm with the photosensitizer to maximum saturation level, intravitreous laser radiation is carried out in parallel light beam of wavelength equal to 661-666 nm is applied at a dose of 30-120 J/cm2. The transformed retina and tumor destruction products are intravitreally removed using vitreotome. Boundary-making endolasercoagulation of retinotomy area is carried out after having smoothed and compressed retina with perfluororganic compound. The operation is finished with placing sutures on sclerotomy and conjunctiva. Platinum, iridium or rhodium are used as the platinum group metals. The number of electrodes is equal to 4-8.

EFFECT: reduced risk of metastasizing.

4 cl, 13 dwg

FIELD: medicine.

SUBSTANCE: method involves building tunnel to posterior eyeball pole in inferoexterior and superexterior quadrants. The tunnel is used for implanting flexible polymer magnetolaser implant to the place, the subretinal neovascular membrane is localized. The implant has a permanent magnet shaped as a cut ring and is provided with drug delivery system and a short focus scattering lens of laser radiator connected to light guide. The permanent implant magnet is axially magnetized and produces permanent magnetic field of 5-7 mTesla units intensity. It is arranged with its north pole turned towards sclera at the place of the subretinal neovascular membrane projection with extrascleral arrangement of laser radiator lens membrane being provided in the subretinal neovascular membrane projection area. The other implant end is sutured to sclera 5-6 mm far from the limb via holes made in advance. The implant is covered with conjunctiva and retention sutures are placed thereon. Light guide and drug supply system lead is attached to temple with any known method applied. Drugs are supplied via the implant drug supply system in retrobulbary way in any order. Triombrast is given in the amount of 0,4-0,6 ml and dexamethasone or dexone in the amount of 0,4-0,6 ml during 3-4 days every 12 h. 0.1-1% aqueous solution of khlorin is intravenously introduced at the third-fourth day after setting the implant as photosensitizer, selected from group containing photolon, radachlorine or photoditazine, at a bolus dose of 0.8-1.1 mg/kg. Visual control of subretinal neovascular membrane cells fluorescence is carried out by applying fluorescent diagnosis methods. After saturating the subretinal neovascular membrane with the photosensitizer to maximum saturation level, intravitreous, transretinal laser radiation of 661-666 nm large wavelength is applied at general dose of 30-120 J/cm2. The flexible polymer magnetolaser implant is removed and sutures are placed on conjunctiva. Permanent magnet of the flexible polymer magnetolaser implant is manufactured from samarium-cobalt, samarium-iron-nitrogen or neodymium-iron-boron system material. The photosensitizer is repeatedly intravenously introduced at the same dose in 2-3 days after the first laser radiation treatment. Visual intraocular neoplasm cells fluorescence control is carried out using fluorescent diagnosis techniques. Maximum level of saturation with the photosensitizer being achieved in the subretinal neovascular membrane via laser light guide and implant lens, repeated laser irradiation of the subretinal neovascular membrane is carried out with radiation dose of 30-60 J/cm2.

EFFECT: accelerated subretinal edema and hemorrhages resorption; regression and obliteration of the subretinal neovascular membrane; prolonged vision function stabilization.

6 cl

FIELD: medicine.

SUBSTANCE: method involves transpupillarily determining size and location of avascular zones and cushion. Conjunctiva and Tenon's capsule incision is carried out in perpendicular to limb 2-3 mm far from it and pocket is formed with spatula in perpendicular to limb in avascular zones and cushion projection area using laser probe set in the formed pocket. Preliminary trans-scleral laser irradiation is applied to the avascular zones and cushion projection area with pulsating and continuous non-collimated beam of laser radiation of low intensity and wavelength of 800-950 nm having mean power of 5-20 mW at probe end during 5-10 min. Photosensibilizer agent is introduced into one of cubital veins and avascular zones and cushion photosensibilizer agent saturation degree is determined by applying photodynamic fluorescent diagnosis method. Maximum avascular zones and cushion saturation level being achieved, trans-scleral scanning laser radiation is applied to the avascular zones and cushion projection area with wavelength of 661-666 nm during 30-90 s with radiation dose being equal to 20-60 J/cm2. The operation is finished without placing sutures over conjunctiva.

EFFECT: enhanced effectiveness of treatment.

2 cl

FIELD: medicine.

SUBSTANCE: method involves applying trans-scleral laser radiation to avascular zones and cushion. Size and location of avascular zones and cushion are transpupillarily determined. Conjunctiva and Tenon's capsule incision is carried out in perpendicular to limb 2-3 mm far from it. Pocket is formed with spatula in parallel to limb in avascular zones and cushion projection area. Photosensibilizer agent is intravenously introduced into one of cubital veins and avascular zones and cushion photosensibilizer agent saturation degree is determined by applying photodynamic fluorescent diagnosis method. Maximum avascular zones and cushion saturation level being achieved, trans-scleral scanning laser radiation is applied to the avascular zones and cushion projection area with wavelength of 661-666 nm during 60-120 s with radiation dose being equal to 40-80 J/cm2. The operation is finished without placing sutures over conjunctiva.

EFFECT: enhanced effectiveness of treatment.

2 cl

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