Photosensitiser and method for production thereof

专利摘要:
FIELD OF THE INVENTION The present invention relates to medicines and relates to photosensitizers for the detection and treatment of tumors. The photosensitizer of the invention is embodied in the form of a composition containing chlorine in the form of salts and alkali metal salts. 80-90% of chlorine e 6 , 50-20% of Purpurin 5 and 18 of Perpurin 18 are used as chlorine. The photosensitizer is prepared by extracting spirulina or biomass with the help of acetone. The biomass is then exposed to acid treatment, neutralization, hydrolysis, extraction of pheopodides α, dissolution in acetone, addition of strong bases, neutralization, and reprecipitation of chlorine e 6 .
公开号:KR20040025911A
申请号:KR10-2003-7012876
申请日:2001-10-04
公开日:2004-03-26
发明作者:레쉐트니코프안드레이발렌티노비치；잘레브스키이고르드미트리에비치；케모프주리빅토로비치；이바노프안드레이발렌티노비치；카르메니안아르타쉐스바체에비치；그라드주시코알렉산드르티크호노비치；라프테프블라디미르페트로비치；뉴고도바나탈리아페트로브나；아바쿠모바올가유리에브나；프리발로프발레리알렉세에비치；라파알렉산드르블라디미로비치；로마노프블라디미르알렉산드로비치
申请人:오브스체스트보 에스 오그라니체노이 오트베트스트베노스티주 〃라다-파마〃；
IPC主号:

专利说明:

Photosensitizer and its manufacturing method {PHOTOSENSITISER AND METHOD FOR PRODUCTION THEREOF}
[2] Photosensitizers (PSs) are used as therapeutics in PDT and as fluorescent labels in photodynamic diagnostics (PDD).
[3] Mono -L- aspartyl chlorin _{e 6 (mono-L-aspartil} chlorin e 6) tetrasodium salt "Npe6" is known as the PS (US Patent No. 4,977,177).
[4]
[5] The PS is active in PDT.
[6] Its disadvantages include only one of manufacturing difficulties, too fast tumor accumulation and excretion dynamics that reduce the effective exposure time to the tumor, and the many types of tumor destruction mechanisms possible in the PDT process, namely The degree of accumulation in malignant formation (tumor) is relatively low because of the high hydrophily, which activates only the effect on blood vessels.
[7] The lysil chlorin p ₆ trisodium salt "LCP" is known to be PS (US Pat. No. 5,330,741).
[8]
[9] The PS is active in PDT.
[10] The disadvantages are the difficulty in manufacturing and the fact that it is a mixture of 10: 1 ratios of two monoamides with amide groups in positions 13 and 15, respectively, which entails unclear biodistrubution and excretion. can do.
[11] The pheophorbid α sodium salt is known to be PS (US Pat. No. 5,378,835).
[12]
[13] The PS can selectively accumulate in malignant tumors and is active in PDT.
[14] The disadvantage is that they tend to oxidize (chemical instability) when stored in solution, are not completely soluble in water after storage in the solid state, are hydrophoby and, as a result, are excreted slowly outside of the organism, This is accompanied by prolonged photosensitivity of the skin integument.
[15] Chlorin e ₆ derivatives are also known to be PS (US Pat. No. 5,002,962).
[16]
[17] Wherein R is a saturated or unsaturated linear or branched hydrophobic hydrocarbon substituent having 4 to 25 carbon atoms.
[18] PS in which R is hexyl is tropic for malignant tumors and is an effective agent for PDT.
[19] The disadvantages are the difficulty in preparing and purifying the preparation, the high hydrophobicity, and as a result, the low accumulation in the tumor and the low stability of the aqueous drug formulation solution upon storage.
[20] There is a known method for preparing compositions of PS, chlorins as alkali metal salts, which are intended for medical practice. This method extracts the biomass of a plant (flower) into a 2: 1 to 8: 1 mixture of hydrocarbons having 6-12 carbon atoms and alcohols having 2-10 carbon atoms, wherein the chlorophyll solution is obtained. Is evaporated at atmospheric pressure, alcohols with fewer carbon atoms than extract alcohols are added, hydrocarbons are removed by distillation completely from the mixture at atmospheric pressure, and at the boiling point of the alcohol, but at a temperature lower than 120 ° C. Was added slowly (gradually) to the chlorophyll alcohol solution until pH 11.5-11.8, the mixture was cooled, incubated for 4 hours, filtered, extracted with hydrocarbons having 6-12 carbon atoms, The alcohol phase containing the magnesium complexes of chlorine is separated, the alcohol is evaporated at atmospheric pressure, and the residue has a pH of 3.5 Hydrochloric acid is added until the mixture is aged until chlorine is completely precipitated and filtered, the precipitate is dissolved in methanol, the alkali alcohol solution is added until the pH is 8.5, the PS solution is filtered and evaporated under vacuum. (US Pat. No. 3,102,891).
[21] Disadvantages of this method are the need to remove the solvent from the extract at high temperatures and the use of alcohols, especially methanol, which is homogenous isomerization of the E exo cycle and pheophytins and pheophorobies. Leading to the formation of several different types of oxidation products of pheophorbids (K. Hyvarinen, J. Helaja, P. Kurchen, I. Kipelainen, PH Hynninen. H-1 and C-13 NMR spectra of the methanolic allomerization products of 13 (2)-(R) -chlorophyll a. // Magnetic Resonance in Chemistry.- 1996,-V.33.-N8.-p.646-656), which is defined as It also leads to complex mixtures with compositions that are difficult to manufacture again.
[22] There is a known method of preparing PS, i.e., chlorine e ₆ sodium salt, which adds 1N NaOH solution to a tetrahydrofuran solution of chlorine e ₆ trimethyl ether and stirs the mixture for 2 days at room temperature under nitrogen atmosphere, Water was added followed by extraction of the organic solvent with methylene chloride and bubbling nitrogen gas into the chlorine e ₆ salt solution to remove methylene chloride (US Pat. No. 5,002,962).
[23] Disadvantages of this method are the low availability of a sufficient amount of chlorine e ₆ trimethyl ether, long PS production times due to the chemical inertness of the ester radical at position 13 of the tetrapyrrole macrocycle. , And PS pharmaceutical formulations due to incomplete saponification of the ester groups at position 13 of the macrocycle when stored in the form of aqueous solutions are unstable.
[24] Known methods of preparing PS, ie "LCP" photosensitizers (trisodium salts of Lysyl-Chlorin p ₆ ) for photodynamic therapy, consist of: The biomass is treated 2-3 times with acetone to extract chlorophyll a, the biomass is filtered or centrifuged, the extract is evaporated, the magnesium ions are removed from the chlorophyll molecule and acid is removed to hydrolyze the phthalyl ester group. Treatment, and at the same time methyl alcohol is added, the reaction mass is treated with water, the pheophovida α derivative is extracted with chloromethylene, the extract is neutralized, washed with water, evaporated and alumina Chromatography on, crystallization of methylfefofovid α from a mixture of chloromethylene and methanol, reaction of the resulting pheopovid α derivative with a strong inorganic base in pyridine-diethylether-n-propanol in the presence of oxygen, The reaction mass is treated with water, the aqueous layer is acidified until pH 4, "anxiety Extract "unstable chlorin" with chloromethylene, evaporate the extract, dissolve "unstable chlorine" in tetrahydrofuran again, evaporate the solution, and increase this procedure at 700 nm Continue until it stops, dissolve the obtained purpurine 18 in tetrahydrofuran, esterify with diazomethane, mix the purpurine 18 methyl ester with aqueous solution of lysine in chloromethylene in the presence of pyridine, and mix the mixture Stir at room temperature for 12 hours, remove the solvent in high vacuum, then purify the obtained crude product with reverse phase high performance liquid chromatography (HPLC), remove solvent by lyophilisation, and remove PS To obtain an injection solution for PDT, dissolve in phosphate buffer solution, add 0.1N NaOH solution, and adjust pH 0.1 N HCl is used to adjust the physiological value to pH 7.35 and the solution is filtered through a microporous filter (US Pat. No. 5,330,741).
[25] The disadvantages of this method are low reproducibility, troublesomeness (high vacuum, crystallization, use of column chromatography and HPLC, long reaction times with lysine), and highly toxic and flammable reagents (diazomethane, pyridine, methanol, tetrahydro Pure, diethyl ether). This drawback makes this method unsuitable for the pharmaceutical industry. In addition, the water-soluble target product obtained is only stable for 24 hours in the dark at 4 ° C. in the aqueous state, and in the dark at 4 ° C. in the solid state, which should be stable for at least 6 months according to the requirements of the pharmacopoeia. Stable for only 4 months (Leach MW, Higgins RJ, Boggan JE, Lee S.-J., Autry S., Smith KM Effectiveness of a Lysylchlorin p ₆ / Chlorin p ₆ mixture in Photodynamic Therapy of the Subcutaneous 9L Glioma in the Rat Cancer Research 1992, V. 52, 1235-1239). In addition, in chemical composition, this PS is a mixture of approximately 10: 1 ratios of monoamides at positions 13 and 15, which can lead to unclear biological distribution and excretion in the organism.
[1] The present invention relates to the field of medicine, in particular the field of photodynamic therapy (PDT) using biologically active compounds.
[104] Figure 1 relates to a method according to the invention, which shows the production of Perpurin 5 with temperature when incubated for 30 days.
[105] Figure 2 shows the dependence of the Purpurin 5 content on incubation time at 70 ° C.
[106] 3 shows the dependence of the Purpurin 5 content on incubation time at 100 ° C. FIG.
[107] FIG. 4 shows the drug of “liquid extract of chlorine” material when the pharmaceutical formulation “Radachlorin, 0.5% solution for injection” (“Photochlorin”) is injected intravenously in tumor mice at a dose of 20 mg / kg. It describes kinetics.
[108] FIG. 5A shows the presence of the metabolite of chlorine e ₆ (Formula I), ie Perpurin 5 (Formula II), in which the curve labeled “2” is for PS in blood.
[109] 5B confirms that chlorine e ₆ (formula I) is metabolized to Perpurin 5 (formula II) in the liver.
[110] 6 shows the PMR spectrum of the "liquid extract of chlorine" obtained in Example 2. FIG.
[111] 7 shows the mass spectrum of the "liquid extract of chlorine" obtained in Example 2. FIG.
[112] Figure 8 shows the visible light absorption spectrum of the "liquid extract of chlorine" obtained in Example 2, which is measured in the ethanol solution, the concentration of the material is 5 mkg / ml.
[113] 9 shows the PMR spectrum of chlorine e ₆ .
[114] 10 shows the mass spectrum of chlorine e ₆ .
[115] Figure 11 shows the visible light absorption spectrum of chlorine e ₆ , which is measured in the ethanol solution, the concentration of chlorine e ₆ is 15 mkg / ml (Sore band-5 mkg / ml).
[116] 12 is the PMR spectrum of Perpurin 5.
[117] 13 is the mass spectrum of Perpurin 5.
[118] Figure 14 shows the visible light absorption spectrum of Perpurin 5, which was measured in the ethanol solution, the concentration of Perpurin 5 is 15 mkg / ml (Sore band-5 mkg / ml).
[119] 15 is a PMR spectrum of Perpurine 5 dimethyl ester.
[120] 16 is the mass spectrum of Perpurine 5 dimethyl ester.
[121] Figure 17 shows the visible light absorption spectrum of the Purpurin 5 dimethyl ester, which was measured in the ethanol solution, the Purpurin 5 DME is 15 mkg / ml (Sore band-5 mkg / ml).
[26] It is an object of the present invention to provide pharmaceutical formulations in the form of aqueous solutions with easy preparation separation and purification, harmonized hydrophobic-hydrophilicity, and consequently optimized tumor accumulation rates, complete excretion of tumors and organisms, and high stability upon storage. It is to obtain a PS characterized in that.
[27] This object is achieved by preparing a PS comprising chlorine in the alkali metal salt state, said chlorine being chlorine e ₆ (13-carboxy-17- [2-carboxyethyl] -15-carboxymethyl-17,18 -Trans-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylporphyrin) 80-90%,
[28]
[29] Perpurine 5 (13-carboxy-17- [2-carboxyethyl] -15-formyl-17,18-trans-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylporphyrin) 5-20%, and
[30]
[31] The remainder is purpurine 18 -chlorine p ₆ (13-carboxy-17- [2-carboxyethyl] -15-carboxy-17,18-trans-dihydro-3-vinyl-8-ethyl-2,7,12 , 18-tetramethylporphyrin),
[32]
[33] The components form a composition, and sodium and potassium can be used as the alkali metal.
[34] It is also an object of the present invention to fully achieve the physicochemical and biological properties of the PS, which provides its effectiveness in PDT, as well as the high reproducibility and simplicity of the PS manufacturing process, and Achieve chemical stability for more than one year of medical forms, and to avoid the use of toxic agents.
[35] The gist of the PS manufacturing method according to the present invention is as follows. Spirulina (Spirulina) processes the biomass with acetone till chlorophyll a is completely extracted, filtered biomass, or centrifugation, and treated with acid to remove the magnesium ion to the extract from chlorophyll molecule and to neutralize the extract , Filtered precipitated pheophytin α, and then hydrochloric acid-acetone consisting of 6-16 ml of acetone, 0.6-6 ml of hexane and 5-10 ml of concentrated hydrochloric acid for 1 g of crude pheophytin α Hydrolyze the pheophytin α using the -hexane mixture, stir the mixture for 20 minutes-1 hour while heating to 40-60 ° C., then add hexane (6-16 ml), and add the organic layer of acetone and hydrochloric acid. Wash with a mixture (2-10: 1), wash the aqueous layer with hexanes, and then neutralize the aqueous layer containing pheophovida with excess sodium citrate (tri-, di- or mono substituted) aqueous solution, Precipitated Pheophovid α is filtered off, washed with water, recrystallized from an acetone-water mixture and dried in air until constant weight. Then, the pheopovid α is dissolved in acetone, an aqueous solution of inorganic strong base at a concentration of 0.05-1.00% is added, stirred at 30-60 ° C. for 5-30 minutes, and an aqueous solution of inorganic strong base at a concentration of 1-50% is further added. The mixture was then heated at 40-60 ° C. for 20-90 minutes, neutralized with dilute hydrochloric acid, the chlorine e ₆ precipitate was separated by centrifugation, washed with distilled water until the acid reaction disappeared, 55-80% Gives chlorine e ₆ . Then, chlorine e ₆ was recrystallized from acetone to be filtered off to separate linear tetrapyrroles, washed with distilled water, and chlorine e ₆ was placed in a sealed reservoir at a temperature of 40-100 ° C. Time-heated for 30 days, then cooled, strong base solution is added until pH 7.5-8.5, and the resulting solution is adjusted with apyrogenic water for injections to adjust the concentration of the photosensitizer to 6.5-7.5 It is made by mass%.
[36] In addition, in the process for preparing PS, the gel filtrated the mixture after the step of adding a strong base until pH 7.5-8.5 to increase the percentage of chlorine e ₆ to 80-90%, Perpurin 5 To 5-20%, the remainder to Purpurin 18 , then add dilute hydrochloric acid solution until the photosensitizer is precipitated, adjust the solution with aspirogen water for injection to make the photoresist concentration 6.5-7.5% by weight. "Liquid extract of chlorins" can be obtained.
[37] Furthermore, in the process for the preparation of PS, after the gel filtration step, dilute hydrochloric acid solution is added until the photosensitizer is precipitated, and then the precipitate is separated by filtration or centrifugation and certified by the RF State Pharmacopeia. Additives can be added until pH 7.5-8.5, injectable apirose water can be added to make the photosensitizer concentration 0.1-1% by weight, and the bacteria can then be filtered off.
[38] In addition, in the preparation method of PS, after the gel filtration step, dilute hydrochloric acid solution is added to the mixture until the photosensitive agent is precipitated, and the precipitate is separated by filtration or centrifugation, and adjusted with apiogen water for injection to adjust the photosensitive agent concentration. 6.5-7.5% by weight, the obtained "liquid extract of chlorine" 0.5-12% by weight is said "liquid extract of chlorine", 5-20% by weight is dimethylsulfoxide, the remainder is water, in the Pharmacopoeia of the Russian Federation It may be dispersed in the gel substrate according to the ratio of the approved additive and the gel substrate.
[39] Furthermore, in the method of preparing PS, after the gel filtration step, dilute hydrochloric acid solution is added to the mixture until the photosensitive agent precipitates, and the precipitate is separated by filtration or centrifugation, and adjusted with apirosen water for injection to adjust the photosensitive agent concentration. To 6.5-7.5% by weight, and the "liquid extract of chlorine" obtained here can be dissolved in dimethylsulfoxide at a rate such that 0.5-12% by weight is "liquid extract of chlorine" and the remainder is dimethylsulfoxide.
[40] The method according to the present invention uses a standard laboratory chemistry pilot equipment to process biomass in a 10-50 liter aluminum vessel equipped with a mechanical stirrer, and the biomass is pumped into an oil vacuum pump and a liquid nitrogen-cooled trap ( Filter through 5-20 liters of nutch filter with trap and centrifuge biomass at rotation speed of 6000 rpm in floor centrifuge to be cooled with 4 x 1 liter buckets Separate, acidify the extract in a 20 liter glass bottle, filter the precipitated pheophytin a through 5-10 liters of a notch filter with an oil vacuum pump and a liquid nitrogen-cooling trap, and filter the pheophytin a Hydrolyze in 0.1-0.5 liter heated three-necked round bottom flask equipped with stirrer, backflow condenser and feeding hole and solution in 2 liter separatory funnel Washed, neutralized in a 2-5 liter beaker, filtered through a 2-5 liter notch filter with an oil vacuum pump and a liquid nitrogen-cooled trap and filtered through a 0.25-1 liter flat bottom flask (chemical recrystallized in flat-bottom flasks and dissolved in acetone in a 0.5-2 liter heated three-neck round bottom flask equipped with a stirrer, countercurrent cooler and a feeding hole. React with an inorganic strong base, separate chlorine e ₆ precipitate at a rotational speed of 6000 rpm in a cooled floor centrifuge with a 4 x 0.5 liter bucket, and remove chlorine e _{6 in a} 0.25-0.5 liter, 2-5 liter flat bottom flask. recrystallization from and chlorin e ₆ the oil vacuum pump and liquid nitrogen-was passed through a 1-2 l notch filter having a cold trap filtered, heat-resistant glass of 0.05-0.1 liters chlorin e ₆ round bottom Plastic Heated in a flask, reacted with a strong base solution with adjustment in a 0.1-1 liter beaker using a standard pH meter and spectrophotometer, and the mixture was gel filtered on a column having a 50-10 mm diameter and 100-150 mm height Bacteria are filtered off through a standard 0.22 μm micropore filter of the Millipore type, and the “liquid extract of chlorine” is dispersed in the gel substrate using a cutter or bead homogenizer. In a conical flask of 0.01-10 liters, a 0.005-2 liter cylinder, a beaker of 0.05-2 liters, a bottle of 20 liters, a weigher in the range of 1-1000 g, and Magnetic stirrers are used for the preparation of samples and solutions; A 5 liter round bottom flask equipped with a thermometer and a direct-flow water condenser is used for the regeneration of acetone and hexane; Rotary vacuum evaporators are used to quickly remove solvent at low temperatures.
[41] In the process for preparing PS, the concentrated hydrochloric acid solution is considered to be a saturated aqueous solution of hydrogen chloride containing typically 36-37% by weight of hydrogen chloride at a temperature of 20 ° C.
[42] In the step of converting pheophytin α to pheopovid α, the volume range of hexane and acetone (6-16 ml of acetone and 0.6-6 ml of hexane) is used to reduce the volume of pheopi when the volume of solvent used is smaller than this. This is explained by the fact that tin α is not completely dissolved and if the volume is larger than this, the solution will not be thick enough to rapid hydrolysis. The volume range of hydrochloric acid (5-10 ml) decreases the yield of pheophobide α when the volume is smaller than this, and the reaction selectivity due to the production of pyrofeopovid α, which is a by-product when the volume is larger than this. This is explained by the fact that it is lowered. The temperature range of 40-60 ° C. decreases the yield of pheophovida α at lower temperatures, and lowers the selectivity of the reaction due to the formation of by-product pyofopevidide α at higher temperatures. Because. The time range of 20 minutes-1 hour indicates that the yield of pheophovida α decreases when the time is shorter, and the selectivity of the reaction is due to the generation of pyrofeopovida α, which is a by-product when the time is longer than this. Because it is lowered. The volume of hexane added (6-16 ml) is not effective to separate phytol, which is one of the products from the reaction mass when the volume is smaller, and it is not reasonable to use a lower volume of hexane. This is explained by the fact that it is not.
[43] In the purification step of the pheopodide α, the organic layer is washed with a mixture of acetone and concentrated hydrochloric acid in a ratio of 2: 1 to 10: 1. If the mixing ratio is lower than 2: 1, a flaky admixture precipitate is produced, which makes it difficult to separate it from the aqueous layer containing the target pheophovidide α. If this ratio is greater than 10: 1, the aqueous layer is supersaturated with acetone, so that a mixture with the hexane layer is formed to contaminate the target pheophobide a.
[44] In the step of converting pheopovid α to chlorine e ₆ , the concentration of the strong base is in the range of 0.05 to 1.00%, the lower limit being the minimum value necessary for the ring-opening reaction of the cyclopentanone ring (E ring) of pheopovid α, If the concentration of the base is greater than 1%, homozygous (oxidation) reaction of the E ring occurs to produce "unstable chlorine" instead of the target chlorine e ₆ , followed by purpurine 18 and further to chlorine p ₆ .
[45] Then, in the process according to the invention, an additional volume of inorganic strong base is added in an aqueous solution at a concentration of 1-50%. If this concentration is lower than 1%, hydrolysis of the ester group at position 13 or 15 occurs incompletely. When the concentration of the base is higher than 50%, the tetrapyrrole macro ring of PS may be ring-opened.
[46] The reaction mass is then stirred at 30-60 ° C. for 5-30 minutes, with lower temperatures facilitating the process of homomorphization of the E ring, with higher temperatures degrading chlorine e ₆ to chlorine e ₄ . Make it easy to be. The temperature range when adding an additional volume of inorganic strong base is 40-60 ° C. and the time range is 20-90 minutes. If the time and temperature are less than this, the methyl ester at position 15 ² is not hydrolyzed, and if the time and temperature are greater than this, the yield of by-product chlorine e ₄ increases.
[47] When converting chlorine e ₆ into a "liquid extract of chlorine", a thermolytic process, which is a dehydration and decarboxylation reaction in which the PS followed by oxidation with the methylene group oxidized at position 15 ¹ is converted to perpurine 5 This happens.
[48]
[49] When converting chlorine e ₆ to "liquid extract of chlorine", if the temperature is lower than 40 ° C, a long reaction time is required, which is not technically reasonable. Temperatures above 100 ° C. result in accelerated decomposition of the material.
[50] A duration of the process of less than one hour requires a temperature of at least 100 ° C., or produces a material with low biological activity.
[51] Process times longer than 30 days entail irreversible change (decomposition) of the material.
[52] The optimum process temperature is 45-70 ° C. (FIG. 1).
[53] The optimal process time is 2-9 days at 70 ° C. (FIG. 2) or 1-48 hours at 100 ° C. (FIG. 3), in which case 5-20% of Perpurin 5 is produced in the mixture.
[54] In the composition of the active agent (PS), substances containing 5-20% Perpurin 5 and 80-95% Chlorin e ₆ are suitable for the preparation of water-soluble injectable pharmaceutical formulations. If the substance contains less than 5% Perpurin 5, the biological activity is low. If the material contains more than 20% of Purpurin 5, the solubility in water will worsen, which adversely affects the stability of the pharmaceutical formulation upon storage and degrades the filtration power through the microporous filter. Since the tetrapyrrole pharmaceutical formulation is not sterilized by heating or UV irradiation because of the high possibility of undesirable chemical change, the latter property is essential for the sterilization of the pharmaceutical formulation.
[55] The 80-95% chlorine e ₆ content in the material is essential to keep the Purpurine 5 water soluble.
[56] The pH range is derived from the fact that its small value, namely pH 7.5, is the lower limit such that the solubility of chlorine in aqueous solution is such that the concentration is suitable for medical use without the addition of stabilizers. The upper limit value, that is, pH 8.5 are hydroxide ions (OH ^-) is a biologically acceptable limits for.
[57] Chlorin e ₆ concentration range of 6.5-7.5% is as having been derived from the use of technological methods of centrifugation or filtration at the stage of separating chlorin e ₆ precipitate, these methods provide a product with a range of concentrations.
[58] PS is described in Example 1, an embodiment of the method is provided in Examples 2 and 3, and special cases of method realization are described in Examples 4-9.
[59] From a chemical point of view, the PS is an alkaline medium (with hydrogenated D ring) and an alkaline medium (when stored) in the three original ring tetrapyrrole-chlorine e ₆ (formula I), perpurin 5 (formula II, Example 10) and (when stored). Perpurin 18 (formula III)-gradually being converted to chlorine p ₆ within.
[60] From a physicochemical point of view, PS has the ability to absorb light in the visible region, which is excited with PS photoactivation and subsequent transfer of energy to organic substrates and molecular oxygen dissolved in tissues. causes relaxation of the state. Such delivery results in oxidation and free radical reactions in living tissue and damage and subsequent destruction (necrosis) of the tissue. The most preferred excitation band for PDT is the long wavelength band (Table 1), because the transmission of light in living tissue increases with increasing wavelength. Thus, PS can damage biological objects up to 10 mm deep after excitation by light with a wavelength of 654-670 nm.
[61] From a pharmaceutical point of view, PS is a "liquid extract of chlorine" substance (extract is considered liquid when the concentration of the active agent is less than 20%). The material is regarded as an extract because it is necessary to extract the biomass from the biomass using an organic solvent. Table 1 shows the absorption maximum position and extinction absorption values of molecules in the long wavelength band of the "liquid extract of chlorine" present in different media.
[62] PS λ _max , nmε, M ^-1 cm ^-1 (0.01M borate buffer, pH 9.18) λ _max , nmε, M ^-1 cm ^-1 (0.01M borate buffer solution containing 1% human serum albumin, pH 7.2) λ _max , nmε, M ^-1 cm ^-1 (ethanol) "Cloline Liquid Extract" 654.5 (28270) 662 (34200) 662 (34230)
[63] Compounds of formula II have the ability to selectively accumulate in malignant neoplasms and infected focuses, but are poorly soluble in water, and compounds of formula I, with apparent photodynamic activity, dissolve for compounds of formula II A solubilizeing agent.
[64] In the pharmacological view (FIG. 4, Example 11), the uniqueness of the pharmacokinetic parameters is that the PS of formula I is gradually converted to the PS of formula II in the organism, and this reaction is introduced into the organism. From the moment of excretion to the excretion of the tumor, by maintaining the concentration of PS of Formula II at a constant level for a time sufficient for PDT to be effectively realized. After injection into the tumor mouse organism, the proposed PS composition enters the blood flow, and the blood concentration is high and stable PS concentration-0.27, mainly by compounds of formula I, sufficient for effective PDT in the 0.5-4 hour range. -0.32 μM − is achieved within the first 3 hours after injection into the tumor area. Due to the presence of 5-20% of the compound of formula (II) in the composition, a high contrast is achieved within this time, which has the ability to accumulate in the tumor as a significant control, so that PS is an animal This is because the maximum accumulation occurs at the first three hours after injection into the organism of animals (the index of contrast is 14.5 in the skin and 2.9 in the muscle). Within this time, the compound of formula (I) is converted into the compound of formula (II) in the organism, providing a high and stable PS concentration in the tumor area in the 3-5 hours after injection, which gradually decreases, up to 18 after injection. It remains therapeutically sufficient by time. The compound of formula (II) is then broken down into a non-toxic product that is excreted through the liver in the organism.
[65] The conversion of chlorine e ₆ of formula (I) to perpurin ₅ of formula (II) is evidenced by the fluorescence spectra of organs and tissue samples of experimental animals (FIG. 5). "Radachlorin, a 0.5% solution for injection"("Photochlorin") pharmaceutical formulation was added to a blood homogenate at a concentration of 10 μM (FIGS. 5A, (1), followed by spectrophotometric analysis. , A fluorescence spectral change in the form of 1.2 times wider and a shift in the fluorescence intensity maximum to the 8 nm long wavelength spectral region were observed, broadening when “Photochlorin” was added at a lower concentration (C = 1 μM). Without this, only a shift in the spectrum is observed, which shows the dose effect in metabolite production (Figures 5a, (2)).
[66] Figure 5a, (3) shows the results of blood homogenates obtained when 3 hours after the injection of "Photochlorin" into the mouse. The most obvious variable here is that the spectrum is 1.5 times wider in the maximum band transitioned to the 4 nm long wavelength spectral region, indicating the presence of a mixture of “photochlorin” and metabolites in the blood assay sample.
[67] When "Photochlorin" was added to a test tube containing liver homogenate at a concentration of 10 μM (FIG. 5b, (1)), the characteristic change in the spectrum first indicated that the maximum fluorescence intensity shifted to the 9 nm long spectral region. will be. No change in the width of the spectrum is observed.
[68] Similar pictures are also observed when "Photochlorin" was added at lower concentrations, C = 1 μM (Figure 5b, (2)).
[69] When analyzing liver tissue homogenate obtained at 3 hours after preparation introduction into the animal, the photospectral analysis of the sample is similar to the previous two figures (Fig. 5b, (3)).
[70] Thus, the data obtained here show that the "Photochlorin" metabolite is present in liver homogenates.
[71] Accumulation of Perpurin 5 in laboratory animal tumors, which is optimal for the photodynamic effect according to selectivity, is observed in the range of 3-18 hours after intravenous or intraperitoneal injection. If it is also necessary that the chlorine material can be circulated in the bloodstream, the optimal time for irradiation is 0.5-4 hours after intravenous infusion. Typically, to conduct PDT with a "liquid extract of chlorine", the interval between formulation infusion and irradiation is 0.5-18 hours.
[72] The biological activity of the pharmaceutical formulation "Radachlorin, 0.5% solution for injection"("Photochlorin") containing 0.5% anhydrous "liquid extract of chlorine" substance is evaluated in vitro and in vivo .
[73] The PS balance associated with amphiphilicity is demonstrated by standard in vitro experiments (Kessel D. Biochemistry, 1977, V.16, p. 3443-3449) (Table 2, Example 12). The PS distribution coefficient in 1-octanol / phosphate buffer solution, pH 7.4 (Cd) is 1.40. This means that the claimed PS dissolves equally well in both the aqueous and lipid layers, which is redistributed from water into complexes containing transport proteins and lipoproteins. Allows rapid penetration into cells and builds up on cytoplasmic intracellular membranes and microsomes, or by penetration into cells by diffusion across these plasma membranes. To prove the lipophily of the PS. Compounds deposited in this manner after laser irradiation release singlet oxygen into the cell and kill the cell.
[74] PS cells with different anticancer activity against different types of cancer cells were cultured in three lines: rat pheochromocytomes PC 12, rat Gasser's ganglion neurinoma RGGN1 and rat liver cancer 27 This is evidenced by the results obtained in vitro experiments using (Hep27) (Table 2, Example 13).
[75] The methods described below are used for the study of dose dependent cytotoxicity (after laser irradiation) and biological "dark" activity of PS.
[76] 1. MTT-test which makes it possible to accurately define the number of living cells after PS treatment and laser irradiation, in order to calculate the cytotoxic and cytophototoxic index of PS. This experiment makes it possible to calculate the dose dependent cytotoxicity and biological "dark" activity of PS (Andrei V. Reshetnickov, Gelii V. Ponomarev, Andrei V. Ivanov, Olga Yu. Abakumova, Tatyana A. Tsvetkova, Artashes V. Karmenyan, Aleksei G. Rebeko, Rudolf Ph. Baum.Novel drug form of chlorin e ₆ // In Optical Method for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy IX,-Edited by TJ Dougherty, Vol. 3909, 124 -129 (2000)).
[77] 2. Determination of the number of cells after staining the monolayer with crystal violet at the end of the experiment. This method is less difficult and less expensive than MTT-testing, and allows the calculation of the cytotoxicity and photocytotoxicity of PS (AE medvedev et al., Biomed. Science, 1990, v. 1, p.261), but crystal violet Is also less accurate because it also stains dead cells.
[78] 3. The relative genotoxic and photogenic phototoxicity of PS is assessed by the degree of inhibition of DNA synthesis in cells. DNA synthesis assays using a standard radiation (radiometric methods, O. Yu. Abakumova , etc., J. Neural. Transm. Suppl. 3, 1998, V.52, p.87), 14 C -thymidine (thymidine) are Calculated by the level of incorporation into the DNA.
[79] All three cell lines studied are highly sensitive to laser irradiation effects after PS treatment (data from MTT-test). Depending on the sensitivity to laser irradiation, the cell lines are classified as RGGN1> PC12> Hep27.
[80] In darkness survival, the prolonged PS effect at 5 μM concentration on cells was 96.5-86.2% for PC-12, 103.7-93.0% for RGGN1 and 109.7-87.9% for Hep27. (MTT-test-Crystal Violet, respectively). Under the same conditions, DNA synthesis in PC-12 cells remained substantially unaffected, with a value of 21.2 and a decrease of 22.2% in Hep27 and RGGN1 cells, respectively. The notable increase in the number of RGGN1 and Hep27 cells in the 5 μM PS effect on cells in the dark would be most likely related to the induction of cell proliferation activity by PS. In general, cytotoxic activity is more common in PS in the absence of irradiation as compared to induction of proliferative activity.
[81] Cell death is observed after laser irradiation to cells treated with PS. Dose dependent photocytotoxic activity of the formulation is detected, which makes it possible to calculate the EC ₅₀ , ie determine the PS concentration at which 50% of the cells die. This data is shown in Table 2. It should be noted that PS with an EC ₅₀ value of less than 20 μM is considered effective for tumor growth inhibition.
[82] When photogenotoxicity was determined after treatment of cells with 5 μM PS and laser irradiation, DNA synthesis in PC-12 cells was significantly reduced (96.5% reduction compared to the control group subjected to irradiation only). DNA synthesis stimulation after laser irradiation at low PS concentrations is observed in Hep27 and RGGN1 cells, which synthesis is significantly reduced when 5 μM RC is present. Notable DNA synthesis stimuli can be explained by the fact that transformed liver and glia cells surviving at low PS concentrations have high DNA synthesis and population regeneration ability.
[83] PS is thus a highly photo cytotoxic agent against different types of tumor cells. For high concentrations (> 5 μM), it is a suitable tumor growth inhibitor even without irradiation. Because of the high photogenic toxicity, PS can be considered a strong tumor growth inhibitor upon irradiation.
[84] PS toxicity was studied in vivo experiments (Example a4). The mean LD ₅₀ value is 210.53 ± 22.2 mg / kg when considered as a weight factor, with a dose (LD ₁₀ ) of 169.87 mg / kg killing 10% of the experimental animals. These experiments may make PS a "low toxicity" material.
[85] PS biodistribution was studied in an in vivo experiment (Example 11). The following distribution mechanism of the compound was observed by injecting PS into the abdominal cavity of mice inoculated with posterior leg muscle T36 embryo carcinoma. After injection the PS enters the blood and then redistributes into the organs and tissues of the animal (Table 3).
[86] As can be seen in Table 3, the tumor accumulation maximum (0.70 μM) is achieved 5 hours after injection at a dose of 40 mg / kg in the abdominal cavity, which is maintained for a long time (18-24 hours) . The tumor concentration at 18 hours after injection is 0.48 μM, which is 1.5 times smaller than the accumulation absolute maximum at high selectivity accumulation. The tumor / muscle tissue ratio is 32 and the tumor / skin ratio is 44.
[87] Tumor accumulation maximum (0.32 μM) is achieved at 0.5 hours after intravenous injection at a dose of 20 mg / kg, which is also maintained for a long time (up to 5 hours). The maximum control in the intravenous injection is achieved at 3 hours, which makes the tumor / muscle tissue ratio 3 and the tumor / skin ratio 4. PS is excreted out of the organism by up to 98% per day. Table 2 below shows the lipophilic coefficient and in vitro activity of “Radachlorin, 0.5% solution for injection” (“Photochlorin”).
[88] Test, cell line Cytotoxicity (“dark” toxicity),% against control at 5 μM Light cytotoxicity, EC ₅₀ , μM ¹ (CD) MTT test, PC-12 96.5 1.8 1.40 MTT Test, RGGN1 103.7 1.8 MTT Test, Hep27 109.7 3.9 Crystal Violet Test, PC-12 86.2 1.5 Crystal Violet Test, RGGN1 93.0 1.8 Crystal Violet Test, Hep27 87.9 4.7 Genotoxicity, PC-12 104.7 3.5% for control at 5 μM Genotoxicity, RGGN1 77.8 132.2% for control at 5 μM Genotoxicity, Hep27 78.8 100.7% for control at 5 μM
[89] ¹ Except photogenic gene toxicity
[90] The results of calculating the effect of the formulation on cancer PDT in in vivo experiments in mice indicate that "Radachlorin, 0.5% solution for injection" ("Photochlorin") and "" Radachlorin, 0.05% gel "have obvious photodynamic activity. Make it possible.
[91] "Liquid extracts of chlorine" pharmaceutical substances, including chlorine sodium salts (or salts of chlorine and other inorganic strong bases), include calcium carbonate, saccharose, glucose, starch, magnesium stearate, polyvinylpyrrolidone, polyglucan For the preparation of pharmaceutical formulations by supplementing other additives approved by the Pharmacopoeia of the Russian Federation, such as methylglucamine, isotonic solution, dimethylsulfoxide, gels and water-emulsion substrates, etc. (Examples 4-9).
[92] Ointments, liniments, gels, oil-based preparations are used externally, and these formulations are approved by the Pharmacopoeia of the Russian Federation, 5-20% dimethylsulfoxide and 0.5-12% It contains a "liquid extract of chlorine" material, or contains 0.8-14% "liquid extract of chlorine" material and 86-99.2% dimethylsulfoxide (Examples 8, 9).
[93] The concentration range of dimethylsulfoxide in combination with the substrate is explained by the fact that when the concentration is lower than 5%, material penetration into the tissue is low, which reduces the PDT effect. If the concentration of dimethylsulfoxide is higher than 20%, pharmaceutical formulations based on other bases lose stability on storage. The concentration range of the substance is explained by the fact that when this concentration is lower than 0.5%, the substance concentration in the tissue is not sufficient for effective PDT. If the substance concentration is higher than 12%, the tissue loses its permeability to light irradiation so that all light is absorbed in the upper layer of the tissue, which causes burns at low efficiency of the PDT procedure. Table 3 below shows the main pharmacokinetic parameters.
[94] PS Absolute accumulation value, long-term-μM-time Tumor accumulation maximum, μM-hour Tumor / skin ratio at tumor accumulation maximum Tumor / Muscle Ratio at Tumor Accumulation Maximum Tumor Accumulation at Control Maximum, μM-Time Tumor / Skin Ratio at Control Maximum Tumor / Muscle Ratio at Control Maximum Excretion,%-hour Phochlo-rine intravenous, 20 mg / kg Collectible-4.0-0.5 0.32-0.5 6.4 1.6 0.29-3 14.5 2.9 98-24 Phochlo-rine intraperitoneal, 40 mg / kg Blood-5.2-0.5 0.70-5 3.9 3.0 0.48-18 44.0 32.0 98-24
[95] For external use, the substance is applied to the skin 0.5-24 hours before irradiation. If this time is less than 0.5 hours, there is no time for the material to penetrate into the tissue of the required depth. If the time interval is longer than 24 hours, it is observed that the absolute accumulation value of the formulation is lowered due to the redistribution and excretion of the formulation. In addition, it is inconvenient from the clinical point of view to apply the external pharmaceutical formulation to the skin for a long time.
[96] The formulation is intended for intravenous drip or stream introduction in the form of 0.1-1% solution dissolved in any of the media approved by the Pharmacopoeia of the Russian Federation (injectable apirose water, dimethylsulfoxide, saline, etc.). Used. If the solution of the substance is used at a concentration of less than 0.1% is unreasonable considering the volume of liquid injected into the organism. It is not possible to use solutions of concentrations higher than 1% because these solutions have low filterability at the stage of sterilization by passing through antibacterial filters.
[97] Semiconductor laser diode module ML-662-SP for photodynamic therapy devised by ZAO "MYLON" (St. Petersburg) and OOO "SIGM PLUS" (Moscow) is used for the activation of the PS "liquid extract of chlorine" material. The module has the following output data (Verification by the Russian Ministry of Health, registration number 29 / 10-679-96):
[98] Power of 2.5-3W in 200 micron fiber with 0.22 aperture.
[99] -High intensity laser diode with a maximum irradiation wavelength of 662 ± 3 nm, co-produced by "Polaroid" (USA) and OOO "SIGM PLUS".
[100] Low power (with a small number of diodes) modules with a maximum irradiation wavelength of 662 ± 3 nm may also be used for PS material activation, yttrium-aluminum garnet YAG: Nd ³⁺ with a maximum irradiation wavelength of 670 nm. Solid-state lasers with pumping on the second harmonic of may be used.
[101] The amount of energy supplied varies from 30 to 3000 J. When irradiating light below 30 J, the PDT procedure takes too long because scanning must be performed in extremely small areas for optimum effect. In tumor dimensions of more than 3000 J and most frequently occur in clinical dimensions, significant damage of healthy tissue is observed, leading to prolongation of the regeneration period.
[102] The surface density of the energy supplied varies from 50 to 2500 J / cm 2. In surface light irradiation below 50 J / cm 2, no effect is observed. In the case of surface light irradiation greater than 2500 J / cm 2, significant damage of healthy tissue is observed, leading to prolongation of the regeneration period.
[103] The wavelength range of the excitation irradiation depends on the technical characteristics of the laser used (662 ± 3 nm), the transition of the preparation absorption maximum value (654-662 nm) and the fur in the material depending on the polarity of the medium. It is related to the content of Purine 5 (5-20%, half-width of the long wavelength absorption band at 663-670 nm (Table 1).
[122] Example 1 . Explanation of Physicochemical Properties of PS
[123] PS is a dense black mass with a thin layer of green shade and algae fragrance.
[124] 7.5% of the "liquid extract of chlorine" is thoroughly stirred to confirm the certainty of the PS properties, and a portion of the extract (1 mg) is 10 ml of medicinal or best purified rectified ethyl alcohol (95%) Dissolve in and measure the optical density at 662 nm (D). The value is 0.23. The molecular extinction value ε (M ⁻¹ cm ⁻¹ ) is calculated according to the equation ε = D * 597 / (0.004). The resulting value should be in the range 33300-35100. Ε = 0.23 * 597 / (0.004) = 34328 after substitution. Thus "liquid extract of chlorine" contains 7.5% PS.
[125] PS ethyl alcohol solution has yellow green color. The solution becomes ruby-red when passing a beam of medical blue lamp MDS 220-75 (technical data 16.535.376-79) through the solution layer in a location where light is blocked.
[126] Agitation of the "liquid extract of chlorine" for quantification, dissolve a portion of the extract (5 mg) in 10 ml of medicinal or best purified rectified ethyl alcohol (95%), at 662 nm Optical density is measured (D). The value is 2.15. PS content is calculated according to the formula c (%) = (D * 596 * 10 * 100) / 34230 * 5). The resulting value should correspond to the specified value. After substitution c (%) = (2.15 * 596 * 10 * 100) / 34230 * 5) = 7.5% (corresponding to the stated value).
[127] For further analysis, add diluted hydrochloric acid solution to 100 mg of "liquid extract of chlorine" until PS precipitates, filter the precipitate, add phosphorus pentoxide and dry under vacuum for 12 hours, PMR, mass spectrum and absorption spectrum Is measured in the wavelength region of 360-720 nm.
[128] PS PMR spectrum (FIG. 6): (DMSO-D6 concentrated solution): 9.64, 9.55, 9.52, 9.39, 8.90, 8.79 (s, meso- H of chlorine e ₆ and perpurin 5), 8.09, 8.04, 7.97, 7.92 (2d, C H = CH ₂ of Chlorin e ₆ and Perpurin 5), 6.84 (s, γ-meso-C H O of Perpurin 5), 6.37, 6.32, 6.13, 6.10 (2d, CH = C H ₂ ), 5.43 (2s, γ-meso-C H ₂ COOH), 4.60 (m, 7- H ), 4.45 (m, 8- H ), 3.80, 3.56 (qx2, 4-C H ₂ CH ₃ ), 3.75 , 3.64, 3.51, 3.46, 3.29, 3.23 (c, nuclear C H ₃ of chlorine e ₆ and Perpurin 5), 2.38, 2.32 (2 m, 7-C H ₂ CH ₂ COOH), 2.71, 2.20 (2 m, 7 -CH ₂ C H ₂ COOH), 1.76 (d, 8-C H ₃ ), 1.72 (t, 4-CH ₂ C H ₃ ), 1.63, 1.91 (2s, 2N H ) ppm.
[129] PS mass spectrum (FIG. 7): ei, 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).
[130] PS visible light absorption spectra: lambda (ε) (ethanol) 386 (22310), 406 (113040), 506 (14870), 536 (8925), 608 (7437), 662 (34220).
[131] According to the PMR spectrum, the substance contains 80% of chlorine e ₆ , 15% of purpurine 5 and 5% of purpurine 18 (small signals at 9.25, 9.10, 8.71, 7.84, 3.55, 3.32, 3.04 ppm). Which corresponds to the claimed composition. According to the mass spectrum, there are peaks of molecular ions 596 of chlorine e ₆ and perpurine 5 molecular ions 566. In the absorption spectrum there is a band of 662 nm, which is an absorption value well suited to the molecular extinction of the PS etalon 34230.
[132] Thus the sample studied was "liquid extract of chlorine", 7.5%.
[133] Example 2. Preparation of "Liquid Extract of Chlorine", 6.5% Phosphorus
[134] Spirulina biomass (2 kg) was treated with acetone (3 x 2 L) until chlorophyll a was completely extracted, the biomass was filtered and the extract was extracted with hydrochloric acid (30 ml) to remove magnesium ions from the chlorophyll molecule. Treatment, neutralize the extract and filter the precipitated pheophytin α (8 g). The pheophytin α is then hydrolyzed in a hydrochloric acid-acetone-hexane mixture, for which the pheophytin α is dissolved in a mixture consisting of 50 ml of acetone, 5 ml of hexane and 40 ml of hydrochloric acid (37%). The mixture was stirred for 1 hour while heating to 40 ° C., then hexane (50 ml) was added, the organic layer was washed with a mixture of acetone and hydrochloric acid (2: 1, 3 × 50 ml), and the aqueous layer was washed with hexane ( 5 x 40 ml). Next, neutralize the aqueous layer containing pheophovida with excess sodium citrate (tri-, di- or mono-substituted) aqueous solution, filter the precipitated pheopovid α and filter with water (3 x 50 ml). ), Recrystallized from an acetone-water mixture and dried in air until constant weight (yield of pheopovid α is 4.2 g, 7.1 mM, 77%). Then, pheopovid α (2.7 g, 4.56 mM) was dissolved in acetone (100 ml), an aqueous inorganic strong base solution (0.05%, 25 ml) was added, stirred at 60 ° C. for 5 minutes, and an aqueous inorganic strong base solution ( 20%, 25 ml) is added further. The mixture is then heated at 40 ° C. for 90 minutes, neutralized with dilute hydrochloric acid (2%, about 250 ml), the chlorine e ₆ precipitate is separated by centrifugation and distilled water (5 × 10 until the acid reaction disappears). ml) to give 1.85 g (2.96 mM, 65%) of chlorine e ₆ . Then, chlorine e ₆ was recrystallized with acetone to separate linear tetrapyrrole, filtered, washed three times with distilled water, chlorine e ₆ was placed in a sealed reservoir and heated at a temperature of 40 ° C. for 30 days, and then cooled. Add 1% sodium hydroxide solution until pH 7.5. The PS obtained here contains 15% Perpurin 5, 80% Chlorin e ₆ and 5% Perpurin 18 (Chlorin p ₆ ). The PS solution is then adjusted with distilled water to make the photosensitizer concentration 6.5%, yielding 14.2 g (50%) of PS in the form of 6.5% "liquid extract of chlorine".
[135] PMR spectrum of “liquid extract of chlorine” obtained here (FIG. 6): (DMSO-D6 concentrated solution): 9.64, 9.55, 9.52, 9.39, 8.90, 8.79 (s, meso- H of chlorine e ₆ and perpurin 5) , 8.09, 8.04, 7.97, 7.92 (2d, C H = CH ₂ of chlorine e ₆ and Perpurin 5), 6.84 (s, γ-meso-C H 0 of Perpurin 5), 6.37, 6.32, 6.13, 6.10 (2d, CH = C H ₂ ), 5.43 (2s, γ-meso-C H ₂ COOH), 4.60 (m, 7- H ), 4.45 (m, 8- H ), 3.80, 3.56 (qx2, 4- C H ₂ CH ₃ ), 3.75, 3.64, 3.51, 3.46, 3.29, 3.23 (c, nuclear C H ₃ of chlorine e ₆ and perpurin 5), 2.38, 2.32 (2m, 7-C H ₂ CH ₂ COOH) , 2.71, 2.20 (2m, 7-CH ₂ C H ₂ COOH), 1.76 (d, 8-C H ₃ ), 1.72 (t, 4-CH ₂ C H ₃ ), 1.63, 1.91 (2s, 2N H ) ppm.
[136] The material contains 80% of chlorine e ₆ , 15% of purpurine 5 and 5% of purpurine 18 (chlorine p ₆ ) (signals at 9.25, 9.10, 8.71, 7.84, 3.55, 3.32, 3.04 ppm). .
[137] Mass spectrum of material obtained (FIG. 7): ei, 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).
[138] Visible light absorption spectrum (FIG. 8): λ (ε) (ethanol) 386 (22320), 406 (113110), 506 (14880), 536 (8930), 608 (7440), 662 (34230).
[139] Example 3. "Liquid Extract of Chlorine", Preparation of PS at 7.5% Status
[140] Spirulina biomass (2 kg) was treated with acetone (3 × 2 L) until chlorophyll a was completely extracted, the biomass was centrifuged, and the extract was hydrochloric acid (30 ml) to remove magnesium ions from the chlorophyll molecule. Treatment, neutralize the extract and filter the precipitated pheophytin α (8 g). The pheophytin α is then hydrolyzed in a hydrochloric acid-acetone-hexane mixture, for which the pheophytin α is dissolved in a mixture consisting of 100 ml acetone, 50 ml hexane and 80 ml hydrochloric acid (37%). The mixture was stirred for 20 minutes while heating to 60 ° C., then hexane (100 ml) was added, the organic layer was washed with a mixture of acetone and hydrochloric acid (5: 1, 3 × 50 ml), and the aqueous layer was washed with hexane ( 5 x 40 ml). Next, neutralize the aqueous layer containing pheophovida with excess sodium citrate (tri-, di- or mono-substituted) aqueous solution, filter the precipitated pheopovid α and filter with water (3 x 50 ml). ), Recrystallized from an acetone-water mixture and dried in air until constant weight (yield is 3.8 g, 6.4 mM, 67%). Then, pheopovide a (2.7 g, 4.56 mM) was dissolved in acetone (100 ml), an aqueous inorganic strong base solution (1%, 25 ml) was added, stirred at 30 ° C. for 30 minutes, and an aqueous inorganic strong base solution ( 20%, 25 ml) is added further. The mixture is then heated at 60 ° C. for 20 minutes, neutralized with dilute hydrochloric acid (2%, about 250 ml), the chlorine e ₆ precipitate is separated by centrifugation and distilled water (5 × 10 until the acid reaction disappears). ml) to give 1.67 g (2.67 mM, 55%) of chlorine e ₆ . Then, chlorine e ₆ was recrystallized with acetone to separate linear tetrapyrrole, filtered, washed three times with distilled water, chlorine e ₆ was placed in a sealed reservoir and heated at a temperature of 100 ° C. for 1 hour, and then cooled. Add 1% potassium hydroxide solution until pH 8.5. The PS obtained here contains 2% perpurine 5, 82% chlorine e ₆ and 16% perpurine 18 (chlorine p ₆ ). The PS solution is then adjusted with distilled water to make the photosensitizer concentration 7.5% to obtain 11.1 g (50%) of PS in the form of 7.5% paste.
[141] The spectrum of the material obtained here is similar to that obtained in Example 2, showing an overlap of the spectrum of chlorine e ₆ (FIGS. 9-11) and the spectrum of Perpurin 5 (FIGS. 12-14).
[142] Example 4 Special Case of PS Preparation— “Liquid Extract of Chlorine”, Preparation of 7.5%
[143] A PS in the form of the 7.5% paste described in the previous example, containing 2% Perpurine 5, 82% Chlorine e ₆ and 16% Perpurin 18 (Chlorine p ₆ ), was 50 mm in diameter and height the 100 mm of Sephadex G10 using 1% potassium hydroxide solution on the column as the eluent, gel until the chlorine is 90% of the content of e _6, the 5% amount of buffer purine 5, the 5% amount of buffer purine 18 Filtered. Dilute hydrochloric acid solution is added until PS precipitates, and PS is adjusted with aspirogen water for injection to bring the photosensitive concentration to 7.5% by weight, giving 6.8 g of "liquid extract of chlorine", 7.5%. See FIG. 8 for the electron spectrum of the product.
[144] Example 5 Specific Examples of PS Preparation-Preparation of "Radachlorin, 0.1% Solution for Injection" Pharmaceutical Formulations
[145] After gel filtration, a diluted hydrochloric acid solution is added to the solution of PS described in Example 4 until PS precipitates, the resulting precipitate is filtered off, and an aqueous solution of injectable apirosene in concentrated sodium hydroxide is added until pH 7.5, Aspirogen water for injection is added to bring the concentration of PS to 0.1% and then bacteria are filtered out of the solution by passing through an antimicrobial "Millipore" micropore filter with pores of 0.22 μm size. 500 ml of solution are obtained here. See FIG. 8 for the electron spectrum of the product.
[146] Example 6 Specific Example of PS Preparation-Preparation of "Radachlorin, 0.5% Solution for Injection" Pharmaceutical Formulations
[147] After gel filtration, a diluted hydrochloric acid solution is added to the solution of PS described in Example 4 until PS precipitates, the resulting precipitate is filtered off, and a concentrated potassium hydroxide solution is added to pH 7 and then pH Under the control of the meter, the pH was adjusted to 8.5 using N-methyl-D-glucamine and the concentration of the photosensitizer was added to 0.5% by weight of aspirogen for injection, followed by the antibacterial "Millipore with pores of 0.22 μm size. The bacteria are removed from the solution by passing through a micro pore filter. 100 ml of solution are obtained here. See FIG. 8 for the electron spectrum of the product.
[148] Example 7 Specific Example of PS Preparation-Preparation of "Radachlorin, 1% Solution for Injection" Pharmaceutical Formulations
[149] After gel filtration, a diluted hydrochloric acid solution is added to the solution of PS described in Example 4 until PS precipitates, the resulting precipitate is filtered off, and concentrated sodium hydroxide solution is added to pH 8.5, followed by injectable apirosene. Water is added to make the concentration of the photosensitizer 1% by weight and then the bacteria are filtered out of the solution by passing through an antimicrobial "Millipore" micropore filter with pores of 0.22 μm size. 50 ml of solution are obtained here. See FIG. 8 for the electron spectrum of the product.
[150] Example 8 Specific Example of PS Preparation-Preparation of "Radachlorin, Gel" Pharmaceutical Formulations
[151] After gel filtration, a diluted hydrochloric acid solution was added to the solution of PS described in Example 4 until PS precipitated, the resulting precipitate was filtered, and adjusted with aspirogen water for injection to bring the concentration of the photosensitizer to 6.5% by weight, and then It is modified as follows.
[152] Variant (a). 0.3 g of Pemulen TR1 or Carbopol 2020 (BF Goodrich, UK) are added to 75 ml of water and 5 g of dimethylsulfoxide at room temperature and stirred for 1 / 4-8 hours. Aqueous alkali solution is added until pH 5. The gel is resuspended in a supplemental "liquid extract of chlorine", 6.5% and water to bring the concentration of chlorine e _{6 in} the resulting gel to 0.05% and vacuum the gel at 10-50 mmHg for 5 minutes. . Get 100 g of gel.
[153] Variant (b). 5 g of dimethylsulfoxide and "of chlorine liquid extract", the 6.5% made the concentration of chlorin e ₆ in the gel obtained was added to 70 ml water with 0.05% Next, the a Aculin 33A (ISP, USA) 15 g of do. The material is stirred and homogenized, and an aqueous alkali solution is added until pH 5. The gel is evacuated at 10-50 mmHg for 5 minutes. Get 100 g of gel.
[154] After gel filtration, a diluted hydrochloric acid solution was added to the solution of PS described in Example 4 until PS precipitated, and the resulting precipitate was filtered and adjusted with aspirogen water for injection to bring the concentration of the photosensitizer to 7.5% by weight, The modification is carried out as follows.
[155] Variant (c). 0.7 g of Pemulen TR1 or Carbopol 2020 (BF Goodrich, UK) are added to 60 ml of water and 20 g of dimethylsulfoxide at room temperature and stirred for 1 / 4-8 hours. Triethanolamine aqueous solution is added until pH 8.5. The gel is suspended in a supplemental "liquid extract of chlorine", 7.5% and water to bring the concentration of chlorine e _{6 in} the resulting gel to 1%, and the gel is evacuated at 10-50 mmHg for 5 minutes. . Get 100 g of gel.
[156] Variant (d). 20 g of dimethylsulfoxide and "liquid extract of chlorine", 7.5% were added to 55 ml of water to make 1% of the concentration of chlorine e _{6 in} the gel, followed by 15 g of Aculin 33A (ISP, USA) do. The material is stirred and homogenized, and an aqueous triethanolamine solution is added until pH 8.5. The gel is evacuated at 10-50 mmHg for 5 minutes. Get 100 g of gel.
[157] Example 9 Specific Example of PS Preparation-Preparation of "Radachlorin, External Use Dimethyl Sulfoxide Solution" Pharmaceutical Formulations
[158] Variant (a). After gel filtration in Example 4, diluted hydrochloric acid solution was added until PS precipitated, the resulting precipitate was filtered and adjusted to aspirogen water for injection to bring the concentration of PS to 7.5% by weight, followed by the obtained "liquid liquid of chlorine" 14 g of the extract "is added to 86 g of dimethylsulfoxide at room temperature to bring the concentration of chlorine e _{6 in} the resulting solution to 1% and homogenize by stirring. 100 g of solution are obtained.
[159] Variant (b). After gel filtration in Example 4, diluted hydrochloric acid solution was added until PS precipitated, the resulting precipitate was filtered and adjusted to aspirogen water for injection to bring the concentration of PS to 7.5% by weight, followed by the obtained "liquid liquid of chlorine" Extract "0.8 g is added to 99.2 g of dimethylsulfoxide at room temperature to bring the concentration of chlorine e _{6 in} the resulting solution to 0.05%, and stirred to homogenize. 100 g of solution are obtained.
[160] Example 10
[161] To identify Perpurin 5, the reaction mixture of Example 2 was gel filtered on a Sephadex G10 column using 1% N-methyl-D-glucamine solution as eluent, and three fractions. fractions are obtained, wherein the first and second fractions contain Perpurin 5. These fractions are neutralized, the precipitate is filtered off and then dissolved in a chloroform-methanol 1: 1 mixture and esterified with diazomethane. The mixture is washed with water, the organic layer is separated, dried over anhydrous magnesium sulfate, concentrated by evaporation under vacuum, chromatographed on Merck, Kieselgel, 0.04-0.063 silica gel, and the final (lowest liquidity) fractions are collected. If necessary, the obtained Purpurine 5 dimethyl ester (10.1% calculated on the basis of the esterified dry reaction mass) is repeatedly chromatographed.
[162] PMR spectrum (FIG. 15): (DMSO-D6 concentrated solution): 9.64, 9.46, 8.82 ( s, meso - H), 6.82 (s, γ- meso -C H O), 6.34, 6.31 , 6.19, 6.16 (2d , -CH = C H ₂ ), 4.54 (m, 7- H ), 4.46 (m, 8- H ), 3.61 (q, 4-C H ₂ CH ₃ ), 4.20, 3.81, 3.57, 3.53, 3.47 ( 5s, -COOC H ₃ and nuclear C H ₃ ), 2.38, 2.35 (2m, 7-C H ₂ CH ₂ COOH), 2.68, 1.85 (2m, 7-CH ₂ C H ₂ COOH), 1.73 (d, 8 -C H ₃ ), 1.70 (t, 4-CH ₂ C H ₃ ) ppm.
[163] Mass spectrum (FIG. 16): ei, 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).
[164] Visible light absorption spectrum (FIG. 17): λ (ε) (chloroform) 408 (117200), 501 (11380), 542 (9830), 617 (6720), 668 (35200).
[165] Perpurine 5 dimethyl ester is dissolved in acetone and concentrated hydrochloric acid (37%) is added in a ratio of 1: 2. The mixture was stirred at 25 ° C. for 2 hours, neutralized, then Perpurin 5 was filtered off, washed with water, dissolved in 10% N-methyl-D-glucamine solution and then 1% N-methyl-D Gel filter on Sephadex G10 column using glucamine solution as eluent, collect and neutralize second fraction, filter precipitate, wash with water, add phosphorus pentoxide and dry until weight is constant, fur Purine 5 (5.2% calculated on the basis of the esterified dry reaction mass) is obtained.
[166] PMR spectrum (FIG. 12): (DMSO-D6 concentrated solution): 9.55, 9.39, 8.79 (s, meso- H ), 8.09, 8.04, 7.97, 7.92 (2d, -C H = CH ₂ ), 6.84 (s, γ-meso-C H O), 6.37, 6.32, 6.13, 6.10 (2d, -CH = C H ₂ ), 4.60 (m, 7- H ), 4.45 (m, 8- H ), 3.55 (q, 4 -C H ₂ CH ₃ ), 3.75, 3.46, 3.23 (s, nuclear C H ₃ ), 2.38, 2.32 (2m, 7-C H ₂ CH ₂ COOH), 2.71, 2.20 (2m, 7-CH ₂ C H ₂ COOH), 1.76 (d, 8-C H ₃ ), 1.72 (t, 4-CH ₂ C H ₃ ) ppm.
[167] Mass spectrum (Fig. 13): ei, M ⁺ (%) 566 (8.2), 494 (100.0), 447 (9.1), 435 (49.6), 421 (12.7), 405 (6.6), 254 (7.1).
[168] Visible light absorption spectrum (FIG. 14): lambda (ε) (ethanol) 408 (116900), 501 (11320), 540 (9790), 615 (6710), 665 (35090).
[169] Example 11 Pharmacokinetics and Metabolism Studies of "Liquid Extracts of Chlorine" Substances and "Radachlorin, 0.5% Solution for Injection" (Photochlorin)
[170] 769.2 mg / kg of the 6.5% “liquid extract of chlorine” material obtained in Example 2 (50 mg / kg calculated on the basis of anhydrous chlorine material) was injected intraperitoneally of Balb / c line mice. Mice were sacrificed three hours after injection (each group consisted of three mice). 100 g of each of the liver, kidney, spleen, lung, small intestine, tumor, and peripheral muscle tissue materials, as well as faeces from the blood, urine, and colon, are sufficiently homogenized in a glass homogenizer. 4 ml saline solution was added. To test physiological fluids (blood, urine), 0.1 ml of each fluid was taken and dissolved in 4 ml of saline solution. The obtained homogenate was studied with a "Perkin-Elmer" fluorescence spectrometer (model MPF-44A).
[171] Studies on the “Radachlorin, 0.5% Injectable Solution” (“Photochlorin”) pharmaceutical formulations show that the organs of mice at the time of sacrifice at 3 hours after injection of the formulation at 50 mg / kg into the abdominal cavity of mice and It was carried out in a similar manner to that for tissue homogenates.
[172] In both cases the maximum fluorescence intensity in liver, small intestine, spleen and kidney tissues shifted to 670 nm (10-12 nm compared to 0.01 M borate buffer pH 9.2, 0.01 containing 1% human serum albumin) 5-6 nm) compared to M borate buffer solution pH 9.2), indicating the metabolism of “Radachlorin, 0.5% solution for injection” (“Photochlorin”) (FIG. 5).
[173] This phenomenon in the fluorescence spectrum seems to be different from the simple broadening of the spectrum due to the hydrophobic effect of the medium and the transition to the long wavelength region (eg after hydrophobic interactions with proteins, fatty proteins). The transition of intensity maxima is observed with or without the slight widening of the bands typical for new compound production. The fluorescence spectrum of Perpurin 5 in 0.01 M borate buffer solution pH 9.2 containing 1% human serum albumin is characterized by the presence of a 670 nm band.
[174] In the skin and tumors, as well as in blood and lung parenchyma, 1.4-1.5-fold spectral broadening was observed at a wavelength of 669 nm, which is "Radachlorin, 0.5% solution for injection" ("Photochlorin") in homogenates. (A protein complex thereof) and a metabolite present.
[175] "Radachlorin, 0.5 for injection" when "Radachlorin, 0.5% solution for injection" ("Photochlorin") is added directly to tubes containing homogenates of intact animal tissues at a concentration of 0.5-1.0 μM. Metabolite of "% solution" ("Photochlorin") is observed in the blood, small intestine, liver, spleen and lung (transfers to the long-wave region without broadening of the spectrum), and is only half-width in the case of skin homogenates. Is observed to increase by 1.15 times, indicating the presence of a mixture of "Photochlorin" metabolites in the sample.
[176] Increasing the concentration of "Radachlorin, 0.5% injectable"("Photochlorin") in organ homogenates to 5-10 μM, substantially the presence of a mixture of chlorine e ₆ and perpurin 5 is recorded in all samples ( Transition of the spectrum to long wavelengths with a half spectral increase of 1.15-1.05 times).
[177] Therefore, the production of metabolites in "Radachlorin, 0.5% injectable solution" ("Photochlorin") added to the homogenate may be considered to depend on the concentration of the preparation and the enzyme activity of the homogenized tissue.
[178] These experiments clearly show that chlorine e ₆ is converted to perpurin 5 in vivo and ex vivo. This conversion is similar to the conversion of chlorine e ₆ to perpurin 5 upon heating.
[179] Example 12 n-octanol / phosphate buffer solution pH 7.4 partition coefficient
[180] 300 ml of n-octanol and 300 ml of phosphate buffer solution pH 7.4 are vortexed for 20 seconds and centrifuged for 10 minutes at 10000 rpm for separation. In the prepared buffer solution (2 ml) and n-octanol (8 ml), 0.1 ml of a PS aliquot with a PS concentration of 5 mg / ml is dissolved and the absorption maximum at 406 nm is measured.
[181] The values of D ^o _c and D ^b _c are obtained, where o is n-octanol, b is a phosphate buffer solution, and c is a control. 2 ml of phosphate buffer solution containing 0.1 ml of PS and 8 ml of n-octanol were vortexed at 20 ° C. for 20 seconds, followed by centrifugation of 10000 rpm for 10 minutes to provide n-octanol / phosphate buffer solution. Allow equilibrium distribution to take place. The optical density of each phase is measured at 406 nm to obtain D ^o and D ^b values, where o is n-octanol and b is phosphate buffer solution.
[182] C _d is calculated according to the following formula,
[183]
[184] In the formula,V ^o Is the volume of octanol (8 ml) taken to determine the equilibrium distribution,V ^o _c Is the volume of octanol saturated with water (8 ml), taken for control determination of aliquot absorption,V ^b Is the volume (2 ml) of buffering solution taken to determine the equilibrium distribution,V ^b _c Is the volume (2 ml) of buffer solution saturated with octanol, taken for controlled measurement of aliquot absorption. The experiment is carried out three times to obtainC _d Average the values.
[185] The result is 1.4 ± 0.3.
[186] Example 13. Determination of phototoxicity (biological activity) and cytotoxicity (toxicity to cells) of "Radachlorin, 0.5% solution for injection"("Photochlorin") pharmaceutical formulation
[187] For this purpose, laminar "Flow Lab" (UK), CO ₂ -incubator "Flow Lab" (UK), multiscan "Bio-Tek Instruments" (USA), mediums And serum “PanEco” (Russia) are used.
[188] In one experiment, one cell line is transferred to two 48-cell plates, one for laser irradiation and the other for "dark" experiments. The next day, the agent is added to cells in a confluent state and the plates are wrapped in black paper to incubate. Formulations with concentrations of 0.1, 0.5, 2.0 and 5.0 μM are studied. Three hours after the addition of the agent, the cells were irradiated with a laser at a radiation dose of 50 J / cm 2, 39 hours later, followed by an MTT-test and ¹⁴ C to assess DNA synthesis. Incubation with thymidine is also performed ("dark" trays are also tested). In all cases, the upper part of the plate is used for the MTT-test, and the lower part is used to measure the number of cells after DNA synthesis and staining with crystal violet. .
[189] The data shown in Table 2 is the average of four parallel experiments.
[190] Example 14 In Vivo Toxicity Study of "Liquid Extract of Chlorine" Substance and "Radachlorin, 0.5% Solution for Injection"("Photochlorin") Pharmaceutical Formulations
[191] Toxicity is studied by intravenously injecting PS into laboratory white mice (bred at the Russian Academy of Medical Sciences, Krukovo) weighing 19-21 g. While maintaining these animals in standard kennel conditions, the Ministry of Health of the USSR Order No. 1179 of October 10, 1983, "About the approval of specifications of forage expenditures for laboratory animals in health protection institutions" Depend on Toxicity is determined by animal death and the average lethal dose-LD ₅₀ -is calculated. The calculation follows the statistical method recommended in State Pharmacopoeia, 11th edition (1.3). Based on the LD ₅₀ , the studied agents are referred to according to the specific class of toxicity by Hodge and Sterner. Addiction reactions are also recorded during the experiment.
[192] Twelve mice (six males and six females) are used for each PS dose to be tested. To determine the LD ₅₀ of PS, dosages of 5, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 mg / kg are used. A solution with a PS concentration of 5 mg / ml is injected intravenously into mice and the dose is changed by varying the volume of PS injected.
[193] The LD ₅₀ value obtained is 210.53 ± 22.2 mg / kg and the LD ₁₀ value is 169.87 mg / kg.
[194] Example 15 In vivo Biological Activity Studies Using “Radachlorin, 0.5% Injectable Solution” (“Photochlorin”) Pharmaceutical Formulations
[195] The photodynamic activity of the “Radachlorin, 0.5% Injectable Solution” (“Photochlorin”) pharmaceutical formulation is studied in mice of the Balb / c line with T36 embryo carcinoma inoculated into the posterior leg muscles. Mice weigh 20-21 g. Irradiation procedures are performed with diode laser ML-662-SP two weeks after tumor inoculation. The skin of the irradiated area is shaved prior to the irradiation procedure.
[196] The formulation is intraperitoneally injected at a dosage of 40 mg / kg, which is sufficient for treatment. To carry out the investigation procedure, mice are anesthetized with ether. Tumor weights in the control and experimental groups varied from 0.9 to up to 1 g at the time of the experiment. Investigate at 5-6 hours after PS injection. All animals except the control group are examined once and then observed for 1 month after the procedure, and the tumor necrosis area and general physiological state are recorded.
[197] The average density of the exposed radiation dose is 150 or 300 J / cm 2.
[198] The best results for complete tumor necrosis were that crusts were formed one week after PDT in the group receiving 300 J / cm 2 light administration, and the crust was 1.5 months after PDT. Falling is observed.
[199] Example 16 Treatment of Basal Cell Skin Cancer with "Radachlorin, a 0.5% Solution for Injection"("Photochlorin") Pharmaceutical Formulations
[200] Basal cell skin cancer is diagnosed in cytologic examination of scrapes. After dilution in 100 ml of 0.9% sterile NaCl saline solution, the formulation is dropwise injected intravenously to a concentration of 0.7 mg / kg based on the weight of the patient. After 2-3 hours, the tumor is irradiated without anesthesia with a surface irradiation dose of 50 J / cm 2 using a diode laser ML-662-SP having a wavelength of 662 nm. Undesirable side effects are not recorded during the formulation and laser irradiation process. At 2 hours after irradiation, dark brown foci with 1-2 cm of surrounding red areas form in the tumor area. At the end of the first day, necrosis forms in the form of a dark brown dried crust (dry crust) in the tumor area. The scab drops at 2-3 weeks, and after 2 weeks complete epithelization of the skin defects in the previous basal cell carcinoma area with excellent cosmetic effects.
[201] Example 17 Treatment of Basal Cell Skin Cancer Using a “Radachlorin, 0.05% Gel” Pharmaceutical Formulation
[202] Basal cell skin cancer is diagnosed by cytological examination of abrasions. The gel is applied in a thin layer over the tumor so as not to touch the healthy part of the skin as much as possible. Irradiate 20-40 minutes after applying the gel. The irradiation procedure is carried out with a diode laser ML-662-SP ("Mylon-Sigm Plus", Russia) with a wavelength of 662 nm. The density of the exposed radiation dose is 2500 J / cm 2. Two hours after irradiation dark brown lesions with 1-2 cm of surrounding red areas form in the tumor area. At one week, necrosis forms in the form of a dark brown dried crust (dry crust) in the tumor area. The scab drops at 2 weeks, and after 2 weeks complete epithelization of the skin defects in the previous basal cell carcinoma area with excellent cosmetic effects.
[203] Example 18 Removal of a Tattoo Using a "Radachlorin, External 0.5% Dimethylsulfoxide Solution" Pharmaceutical Formulation
[204] The solution is applied to the napkin, the napkin is placed on the tattoo, then covered with black paper or a thin aluminum foil and fixed for 30 minutes. Excess solution is removed from the surface with a cotton swab moistened with alcohol. The irradiation procedure is carried out with a diode laser ML-662-SP ("Mylon-Sigm Plus", Russia) with a wavelength of 662 nm, in which the irradiation is carried out along the pattern line to avoid affecting the surrounding tissue. The density of the exposed radiation dose is 120 J / cm 2. One hour after irradiation, skin redness and swelling are observed in the tattoo area. At the end of the second day, a dark brown “picture” is formed with a peripheral red area of size 1 mm. After 2 weeks, necrosis in the tattoo area is formed in the form of a dark brown scab. After 2 weeks, the scab is peeled off with the tattoo. At smaller doses of light the procedure proceeds without necrosis by stain decolorisation, but in this case repetitive work is required. After 6 weeks, a light pink tissue is formed in the tattoo area as a result of PDT, which is slightly different from the surrounding skin, with good cosmetic effects observed.

权利要求:
Claims (8)
[1" claim-type="Currently amended] Chlorine is Chlorine e ₆ (13-carboxy-17- [2-carboxyethyl] -15-carboxymethyl-17,18-trans-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetra Methylporphyrin) 80-90%,

Perpurine 5 (13-carboxy-17- [2-carboxyethyl] -15-formyl-17,18-trans-dihydro-3-vinyl-8-ethyl-2,7,12,18-tetramethylporphyrin) 5-20%, and

Perpurine 18-Chlorine p ₆ (13-carboxy-17- [2-carboxyethyl] -15-carboxy-17,18-trans-dihydro-3-vinyl-8-ethyl-2,7,12,18- Tetramethylporphyrin), the remainder,

A sensitizer comprising chlorine in an alkali metal salt state, wherein said components form a composition.
[2" claim-type="Currently amended] The photosensitive agent according to claim 1, wherein sodium is used as the alkali metal.
[3" claim-type="Currently amended] The photosensitive agent according to claim 1, wherein potassium is used as the alkali metal.
[4" claim-type="Currently amended] Spirulina biomass is treated with acetone until the chlorophyll a is completely extracted, the biomass is filtered or centrifuged, the extract is treated with an acid to remove magnesium ions from the chlorophyll molecule, and the resulting pheophorbide a derivative is obtained. By reacting with an inorganic strong base, the extract is treated with an acid to remove magnesium ions from the chlorophyll molecule, then the extract is neutralized, the precipitated pheophytin α is filtered off, and then the pheophytin α is hydrochloric acid. In acetone-hexane mixture using 6-16 ml of acetone, 0.6-6 ml of hexane and 5-10 ml of concentrated hydrochloric acid per 1 g of crude feopytin α, the mixture was heated to 40-60 ° C. to 20 After stirring for min-1 h, hexane (6-16 ml) is added, the organic layer is washed with a mixture of acetone and hydrochloric acid (2-10: 1), and the aqueous layer is washed with Next, neutralize the aqueous layer containing pheophobide a with excess sodium citrate (tri-, di- or mono substituted) aqueous solution, filter the precipitated pheopovid a, filter with water, wash with acetone-water. Recrystallize from the mixture, dry in air until constant weight, then dissolve pheopovid α in acetone, add an aqueous solution of inorganic strong base at 0.05-1.00% concentration and stir at 30-60 ° C. for 5-30 minutes Add an aqueous solution of inorganic strong base at a concentration of 1-50%, then heat the mixture at 40-60 ° C. for 20-90 minutes, neutralize with dilute hydrochloric acid, separate chlorine e ₆ precipitate by centrifugation, The reaction was washed with distilled water until the reaction disappeared to obtain 55-80% of chlorine e _6, and then chlorine e ₆ was recrystallized from acetone to separate linear tetrapyrrole, filtered, washed with distilled water, and the chlorine e ₆ was sealed. Placed in a reservoir and heated at a temperature of 40-100 ° C. for 1 hour to 30 days, then cooled, a strong base solution was added until pH 7.5-8.5, and the resulting solution was adjusted with aspirogen water for injection to give a photosensitive concentration. Method for producing a photosensitive agent, characterized in that to make 6.5-7.5% by weight.
[5" claim-type="Currently amended] 5. The method of claim 4, wherein the strong base solution is added until pH 7.5-8.5, and then the mixture is gel filtered to increase the percentage of chlorine e ₆ by 80-90%, perpurin 5 by 5-20%, and the remainder After purine 18, dilute hydrochloric acid solution is added until the photosensitizer is precipitated, and the solution is adjusted with aspirogen water for injection to make the photoresist concentration 6.5-7.5% to obtain "liquid extract of chlorine". Manufacturing method.
[6" claim-type="Currently amended] The method according to claim 5, wherein after the gel filtration step, dilute hydrochloric acid solution is added to the photosensitive solution until the photosensitive agent is precipitated, and then the precipitate is separated by filtration or centrifugation and the pH of the Pharmacopoeia until pH 7.5-8.5. The method according to claim 1, wherein the additive is approved by adding an apirosene for injection to make the photosensitizer concentration 0.1-1% by weight, and then the bacteria are filtered out.
[7" claim-type="Currently amended] The method of claim 5, wherein after the gel filtration step, dilute hydrochloric acid solution is added to the photosensitizer solution until the photosensitizer is precipitated, and then the precipitates are separated by filtration or centrifugation, and the injectable apiogen water is added to adjust the photoresist concentration to 6.5- 7.5% by weight, the "liquid extract of chlorine", 0.5-12% by weight of "liquid extract of chlorine", 5-20% by weight of dimethylsulfoxide, the remainder of water, a pharmaceutically acceptable additive And dispersing on the gel substrate according to the ratio of the gel substrate.
[8" claim-type="Currently amended] The method of claim 5, wherein after the gel filtration step, dilute hydrochloric acid solution is added to the photosensitizer solution until the photosensitizer is precipitated, and then the precipitates are separated by filtration or centrifugation, and the injectable apiogen water is added to adjust the photoresist concentration to 6.5- To 7.5 wt%, wherein the "liquid extract of chlorine" obtained here is dissolved in dimethyl sulfoxide according to the ratio of "liquid extract of chlorine" of 0.5-12 wt% and the remainder is dimethyl sulfoxide. .

类似技术:

---

公开号 | 公开日 | 专利标题

---

US8692014B2|2014-04-08|Acid addition salts of 5-aminolevulinic acid or its derivatives

---

O’Connor et al.2009|Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy

---

Kubin et al.2005|Hypericin-the facts about a controversial agent

---

CA2416307C|2013-01-22|Esters of 5-aminolevulinic acid as photosensitizing agents in photochemotherapy

---

CN1525857B|2013-06-12|Method for regulation of gene expression in eukaryotic cells using nordihydroguaiaretic acid derivatives

---

US5330741A|1994-07-19|Long-wavelength water soluble chlorin photosensitizers useful for photodynamic therapy and diagnosis of tumors

---

US4977177A|1990-12-11|Tetrapyrrole polyaminomonocarboxylic acid therapeutic agents

---

US6036941A|2000-03-14|Fluorogenic substrates for diagnosis and photodynamic treatment of tumors

---

Allison et al.1966|Role of lysosomes and of cell membranes in photosensitization

---

KR100883927B1|2009-02-18|Compound

---

ES2335475T3|2010-03-29|Formulation of 5-aminolevulinic acid in non-waterproof dosages.

---

Damoiseau et al.2001|Increase of the photosensitizing efficiency of the Bacteriochlorin a by liposome-incorporation

---

CA1257202A|1989-07-11|Porphyrins and cancer treatment

---

EP0322795B1|1995-09-20|Novel tetrapyrrole aminocarboxylic acids

---

Gomer et al.1979|Determination of [3H]-and [14C] hematoporphyrin derivative distribution in malignant and normal tissue

---

Kessel et al.1997|The role of subcellular localization in initiation of apoptosis by photodynamic therapy

---

CA2215069C|2006-09-12|Esters of 5-aminolevulinic acid as photosensitizing agents in photochemotherapy

---

AU2003282354C1|2009-10-29|Water-soluble anionic bacteriochlorophyll derivatives and their uses

---

DE60213996T2|2007-09-06|Water-soluble porphyrine derivatives for photodynamic therapy, their use and manufacture

---

Mroz et al.2011|Intraperitoneal photodynamic therapy mediated by a fullerene in a mouse model of abdominal dissemination of colon adenocarcinoma

---

US7335684B2|2008-02-26|Esters of 5-aminolevulinic acid as photosensitizing agents in photochemotherapy

---

CN103298804B|2015-04-29|New chlorine e6-folic acid conjugated compound, preparation method thereof, and pharmaceutical composition containing the same for treatment of cancer

---

WO1990012573A1|1990-11-01|Bacteriochlorophyll-a derivatives useful in photodynamic therapy

---

KR20150014534A|2015-02-06|Methods and compositions for treating lung cancer

---

Zhang et al.2016|Switchable PDT for reducing skin photosensitization by a NIR dye inducing self-assembled and photo-disassembled nanoparticles

同族专利:

---

公开号 | 公开日

---

MXPA03008925A|2005-03-07|

---

AU2002212867B2|2007-07-12|

---

NO327751B1|2009-09-14|

---

GB2389531B|2005-03-16|

---

PL206902B1|2010-10-29|

---

US6969765B2|2005-11-29|

---

ZA200308407B|2005-02-23|

---

CN1250294C|2006-04-12|

---

CZ20032940A3|2004-04-14|

---

NO20034344D0|2003-09-29|

---

EP1380295A9|2004-04-14|

---

CN1512881A|2004-07-14|

---

NZ528237A|2006-09-29|

---

US20060003983A1|2006-01-05|

---

HU0400298A2|2004-07-28|

---

HRP20030876B1|2011-10-31|

---

SI1380295T1|2009-10-31|

---

WO2002078694A1|2002-10-10|

---

SK13482003A3|2004-05-04|

---

EP1380295A1|2004-01-14|

---

HU227756B1|2012-02-28|

---

HRP20030876A2|2005-08-31|

---

SK287784B6|2011-09-05|

---

GB0321726D0|2003-10-15|

---

EP1380295B1|2009-03-25|

---

RU2183956C1|2002-06-27|

---

US7550587B2|2009-06-23|

---

CZ304591B6|2014-07-23|

---

US20040147499A1|2004-07-29|

---

PL365102A1|2004-12-27|

---

HU0400298A3|2007-05-29|

---

NO20034344L|2003-11-21|

---

AT426403T|2009-04-15|

---

EP1380295A4|2005-11-30|

---

KR100628548B1|2006-09-26|

---

CA2440650C|2009-09-15|

---

GB2389531A|2003-12-17|

---

CA2440650A1|2002-10-10|

---

DE60138141D1|2009-05-07|

---

BR0116952A|2004-06-01|

---

BG108534A|2005-02-28|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

法律状态:
2001-03-30|Priority to RU2001108397

---

2001-03-30|Priority to RU2001108397/14A

---

2001-10-04|Application filed by 오브스체스트보 에스 오그라니체노이 오트베트스트베노스티주 〃라다-파마〃

---

2001-10-04|Priority to PCT/RU2001/000399

---

2004-03-26|Publication of KR20040025911A

---

2006-09-26|Application granted

---

2006-09-26|Publication of KR100628548B1

优先权:

---

申请号 | 申请日 | 专利标题

---

RU2001108397|2001-03-30|

---

RU2001108397/14A|RU2183956C1|2001-03-30|2001-03-30|Photosensibilizer agent and method for producing it|

---

PCT/RU2001/000399|WO2002078694A1|2001-03-30|2001-10-04|Photosensitiser and method for production thereof|