RU2568597C1 - Photosensitiser and method for producing it - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention is applicable to produce lyophilised trismethyl glucamine salt of chlorine e6, which is used as a high-effective photosensitiser (PS) for the purpose of photodynamic therapy (PDT) of cancer and other new growths of various origins, as well as for fluorescent diagnosing of cancer cells. The method for producing the PDT photosensitiser involves dissolving methylfeoforbide in acetone, processing the produced solution with aqueous alkali NaOH or KOH, and neutralising the reaction mixture with diluted hydrochloric acid, separating precipitated chlorine e6, washing, processing the precipitation with aqueous solution and lyophilising. Implementing the invention enables eliminating pseudocolloidal precipitation, and producing fine and dense precipitation of the photosensitise low in foreign matters.

EFFECT: photosensitiser provides adequate cryostabilisation, reduced length of patient rehabilitation, more effective lysis of involved and tumour tissues.

2 cl, 2 ex

Description

The invention relates to the field of pharmacology, in particular to biologically active compounds of the chlorin series and to a method for their preparation, and can be used to obtain the trismeglumine salt of chlorin e6 in freeze-dried form, which can be used as a highly effective photosensitizer (PS) for photodynamic therapy ( PDT) of cancer and other neoplasms of various genesis, as well as for fluorescence diagnosis of cancer cells.

Most modern PS for PDT cancer and other diseases use chlorin e6 derivatives. Some of them have found a real embodiment in medical practice and are widely used as approved drugs for PDT cancer and other neoplasms. So, for example, from the data of the Register of Medicinal Products of Russia of the Radar Station of the Russian Federation (see www.rlsnet.ru), approved for clinical use, FS PHOTOLON [RU 2152790 C1 of 07.20.2000, Cl. A61K 31/409] (produced by Belmedpreparaty AO) is a freeze-dried porous powder containing trisodium salt of chlorin e6 with a number of side chlorins (see N. A. Isakau, TV Trukhacheva, AL Zhebentyaev, PT Petrov, HPLC study of chlorine e6 and its molecular complex with polyvinylpyrrolidone. Biomedical Chromatogr., (2007), 21, 318-325) and. A. Isakau, T.V. Trukhacheva, P.T. Petrov Isolation and identification of impurities in chlorine e6. J. of Pharmaceutical and Biomedical Analysis, (2007), 45, 20-29) in combination with polyvinylpyrrolidone (PVP). According to the authors, the presence in PHOTOLONA dosage form of a significant amount of PVP helps to improve some characteristics of this drug as a diagnostic tool.

Another FS is RADACHLORIN [RU 2183956 C1, A61K 31/79, dated June 27, 2002, US Pat 7550587 dated June 23, 2009] produced by RADA-PHARMA LLC - is an aqueous solution of a mixture of various chlorins of variable composition, in which the content of the main product is trisodium chlorine salt e6 is 80-90% of the total amount of all chlorins. And finally, FS - PHOTODITAZINE [RU 2276976 C2, A61K 31/409 of 05/27/2006] (manufactured by BETA-GRAND LLC) - is an aqueous solution of a mixture of bis-N-methyl-D-glucamine salt of chlorin e6 and a small amount of others chlorins (not more than 2-4%) with PVP.

Also known FS based on chlorin e6, which is a bismeglumine monosodium salt of chlorin e6, in the form of a freeze-dried form in the presence of maltose as a cryostabilizer [see RU patent No. 2367434 CL, A61K 31/409, dated 09/20/2009]. However, this FS is not used in practical medicine. Special studies have shown that chlorin e6 has the most pronounced photosensitizing effect, and associated impurities, which for the most part easily aggregate in aqueous media, have a lower quantum yield of singlet oxygen generation (see Yu.A. Bely and A.V. Tereshchenko , PL Volodin, A. Kaplan, GV Ponomarev. A comparative study of the photodynamic effects of chlorine-type photosensitizers on the intact retina of experimental animals “Refractive surgery and ophthalmology 2006, 6, No. 2, pp. 55-7). The effectiveness of the drug as FS is determined by the coefficient of tropism or contrast. Therefore, the impurities of concomitant chlorins present in the PHOTOLON and RADACHLORIN preparations and, in part, in PHOTODITAZINE blur a clear line between the cancerous tumor and nearby healthy tissue, which significantly reduces the effectiveness of PDT when working with this drug due to noticeable photodestruction and nearby healthy tissue. Thus, the presence of impurities even of chlorin nature significantly reduces the effectiveness of the drug as a FS. Therefore, the main goal when creating a FS based on chlorine e6 is to obtain the initial chlorine e6 of the highest possible purity with the lowest possible technological problems.

Currently, there are several methods for producing high-purity chlorin e6. One of them consists in the specific processing of “living” chlorella (Chlorella ellipsoidea) through the sequential isolation of pheophytin and its alkaline conversion to the trisodium salt of chlorin e6 with a total yield of up to 1% with a basic substance content of up to 93-98% in the sample. Korean authors have described in detail the method for producing high purity chlorine e6 (US Pat 8349335, January 8, 2013, Cl. 424 / 195.17), from which it follows that only the use of native (“living”) chlorella directly in the places of its industrial production allows to obtain ultimately a highly purified product with a basic substance content of at least 93-98% with a yield of about 1%, based on dry chlorella. The use of dried chlorella leads to a sharp decrease in yield (up to 20 times). The basis of the method for producing chlorin e6 according to this patent consists of several mandatory steps: 1) repeated washing of the “living” chlorella from inorganic salts; 2) the gradual washing of chlorella with aqueous ethanol to remove polar impurities; 3) extraction of chlorophyll a using 100% ethanol; 4) treatment of the chlorophyll alcohol extract IN with hydrochloric acid to pH 2.5 and separation of the precipitated pheophytin by filtration; 5) crystalline trisodium salt of chlorin e6 by filtration chromatographic purification of pheophytin on a column with a neutral alumina using gradient chromatography in a hexane-methylene chloride system; 6) dissolving crystalline pheophytin in acetone, adding IN NaOH to pH 12; keeping the alkaline solution for 12 hours and separation; 7) dissolution of chlorin e6 salt in water, filtration from insoluble impurities and freeze drying of an aqueous solution.

The closest analogue is the photosensitizer and the method for its preparation described in RF patent No. 2523380.

The photosensitizer for PDT in freeze-dried form includes a chlorine salt e6 and a cryostabilizer, while it contains a trismeglumine salt of chlorine e6 as a salt of chlorine e6, and meglumin in a ratio of 1: 0.1-0.2 parts by weight as a cryostabilizer.

A method of obtaining a photosensitizer for PDT involves dissolving methylpheophorbide a in acetone, treating the resulting solution with aqueous alkali (NaOH or KOH), followed by neutralizing the reaction mixture with dilute hydrochloric acid to pH 4.5-5.0, separating the precipitated chlorine e6 precipitate, followed by washing with distilled water, and treating the precipitate with aqueous solution and lyophilization, while the treatment of the solution of methylpheophorbide a in acetone with aqueous alkali is carried out at a temperature of 40-50 ° C, separation of the chlorine e6 precipitate is carried out by filtration loi Celite 545 and chlorin e6 removed with Celite aqueous meglumine solution until the salt concentration of chlorin e6, the corresponding optical density D = 225-235 / 1 ml at the maximum absorption wavelength 655 nm and pH 9.30-9.35 solution.

The disadvantages of the photosensitizer are that meglumine, which is part of it, does not provide full cryostabilization, a photosensitizer of this composition accumulates in damaged and tumor tissues for a long time, which increases the rehabilitation time of patients and does not allow to efficiently lyse damaged and tumor tissue.

The disadvantage of the method of producing a photosensitizer is that the treatment of a solution of methylpheophorbide a in acetone with aqueous alkali is carried out at a temperature of 40-50 ° C, this leads to incomplete hydrolysis of methylpheophorbide with the formation of a large number of impurities.

The disadvantage is that the neutralization of the reaction mixture with dilute hydrochloric acid to a pH of 4.5-5.0 leads to the formation of a pseudocolloidal precipitate for an extended period of time. This entails the capture by the precipitate of a large amount of acetone and its derivatives from the reaction mixture, which cannot be washed thoroughly by subsequent washing with distilled water.

The objective of the invention is the creation of a photosensitizer and a method for its production, which allows to obtain FS with high consumer properties, long shelf life, maximum purity.

The technical result of the invention on a photosensitizer is to ensure full cryostabilization, reducing the rehabilitation of patients, as well as increasing the efficiency of lysis of damaged and tumor tissue.

The technical result of the photosensitizer is achieved by the fact that the photosensitizer for PDT in lyophilized form, including chlorin e6 salt and meglumine as a cryostabilizer, according to the invention, contains chlorine e6 trismeglumine salt as e6 chlorine and additional dextran as cryostabilizer in the following ratio of components in mass percent:

chlorine salt e6 1-27 meglumine 5-50 dextran rest

The use of additional dextran as a cryostabilizer in combination with other components in the stated ratios allows preserving the consumer qualities of the photosensitizer for longer periods of time, while the amount of dextran cannot be less than 23% due to a decrease in the effect of cryostabilization and cannot be more than 94% of due to a decrease in the effectiveness of the photosensitizer. The amount of chlorin e6 salt cannot be less than 1% due to a sharp decrease in the therapeutic effect of the photosensitizer and cannot be more than 27% due to the high phototoxicity of the photosensitizer.

Meglumin cannot be less than 5% due to a decrease in the effect of cryostabilization and cannot be more than 50% because of a decrease in the effectiveness of the photosensitizer.

The technical result of the invention by a method for producing a photosensitizer for PDT is the elimination of the formation of a pseudocolloidal precipitate, the preparation of a fine, dense sediment of a photosensitizer with a minimum amount of impurities.

The technical result of the method for producing a photosensitizer is achieved by the fact that a method for producing a photosensitizer for PDT, including dissolving methylpheophorbide a in acetone, treating the resulting solution with aqueous alkali NaOH or KOH, followed by neutralizing the reaction mixture with dilute hydrochloric acid, separating the precipitated chlorine e6 precipitate, followed by washing, processing precipitation with an aqueous solution and lyophilization, according to the invention, the treatment of a solution of methylpheophorbide a in acetone with aqueous alkali is carried out at a temperature re 51-60 ° C, neutralization of the reaction mixture with dilute hydrochloric acid is carried out to a pH of 4.0-4.5, separation of the chlorine e6 precipitate is carried out by filtration through a celite layer 545, washing of the chlorine e6 precipitate is carried out with deionized water, then chlorine e6 is extracted from celite with water a solution of meglumine and dextran to achieve a concentration of chlorin e6 salt in the solution corresponding to an optical density of D = 225-235 / 1 ml at a wavelength of absorption maximum of 661 nm and a solution pH of 9.30-9.35.

Treatment of a solution of methylpheophorbide a in acetone with aqueous alkali at a temperature of 51-60 ° C allows hydrolysis to form a minimum amount of impurities. An increase in temperature above 60 ° C can lead to oxidation of the components of the reaction mixture and the formation of additional impurities.

The use of deionized water for washing the precipitate from impurities allows for complete washing of the precipitate with a smaller volume of water with less loss of photosensitizer. The neutralization of the reaction mixture with dilute hydrochloric acid to a pH of 4.0-4.5 allows you to avoid the formation of a pseudocolloidal precipitate, to obtain a fine, dense sediment photosensitizer with a minimum amount of impurities.

A method for producing PS for PDT involves dissolving methylpheophorbide a in acetone, treating the resulting solution with aqueous alkali, followed by neutralizing the reaction mixture diluted with hydrochloric acid to pH = 4.0-4.5, separating the precipitated chlorine e6 precipitate, followed by washing with deionized water, treating the precipitate an aqueous solution of meglumine and dextran and lyophilization. According to the invention, the treatment of a solution of methylpheophorbide a in acetone with aqueous alkali is carried out only at 51-60 ° C, separation of the chlorine e6 precipitate is carried out by filtration through a layer of celite 545 and chlorin e6 is extracted from celite 545 with an aqueous solution of meglumine and dextran until the concentration of chlorin salt is reached in the solution e6, corresponding to an optical density of 230-240 1 ml at a wavelength of maximum absorption of 661 nm and a solution pH of 9.30-9.35, the solution is filtered through 0.22 mmk millipores and lyophilized.

From the data of high pressure liquid chromatography (HPLC) it follows that the destruction of the photosensitizer during 2 years of storage practically does not occur (see Example 1)

Thus, as a result of the proposed treatment of the reaction mixture, a practically non-degradable dosage form is obtained during storage. The decrease in the optical density of the solution over 2 years was only 0.1%, which is many times higher than the stability of all currently known drugs of the FS class.

Example 1

To a solution of 4 g of methylpheophorbide a in 800 ml of acetone, 500 ml of a 10% sodium hydroxide solution was added during 1 hour with intensive argon sparging, stirred for 2 hours at 55 degrees, cooled to 5-10 ° C, 2 l of distilled water was added , the solution is neutralized with vigorous stirring by bubbling argon with dilute hydrochloric acid (1: 3) to a pH of 4-4.5 until an amorphous loose precipitate precipitates so that the supernatant acquires a clear pale violet color, the supernatant is decanted, the precipitate is suspended in a minimal amount of water and filtered through a 5 cm-high celite 545 layer on a glass filter with a diameter of 8 cm, rinse the celite with deionized water until there are no inorganic salts in the filtrate, separate the upper layer of celite 545 containing chlorin e6, place in a flask, add 150 ml 1 % solution of meglumine and 5% solution of dextran in pyrogen-free water, stirred for 5 minutes, the chlorine e6 solution is filtered off, another 250 ml of 1% solution of meglumine and 5% solution of dextran in pyrogen-free water are passed through celite, the celite is further washed further 600 ml of pyrogen-free water, the solution is filtered twice through Stericap filters - 0.22 mmk millipores, the total volume of the solution is adjusted to 1 l, if necessary, the pH of the solution is adjusted to 9.25-9.30, poured into 10 ml dark glass vials, lyophilized under standard conditions and obtained 100 photosensitizer bottles in the form of porous tablets containing in each bottle 37.5-40 mg of the main substance - chlorin e6 (or at least 75-80 mg of heliochlorin). The optical density of the PS for heliochlorin in a vial at 655 nm and a solution pH of 9.0–9.5 is 225–235. Similarly, for PHOTODITAZINE, the optical density D (655 nm) is 205-210 / 1 ml, and for RADACHLORIN, 175-185 / 1 ml.

Example 2

Terms of HPLC:

Chromatograph LC-20 Prominence SHIMADZU (Japan) Pump LC-20AD;

Thermostat STO-20A;

Detector - spectrophotometer SPD-20A;

Column "LUNA" of the company "PHENOMENEX" S-18, 4 × 250 mm, 5 microns;

Eluent MeOH: H2O: CF3COOH (90: 10: 0.01 v / v);

Flow rate - 0.85 ml / min;

Loop volume - 5 μl;

Temperature - 25 ° C;

Detection at 405 nm.

10 μl of a solution of chlorin e6 is diluted in 1 ml of eluent (until an acidic solution is formed, which is determined by a color change from brown to blue, i.e., the appearance of a chlorin e6 dication). The resulting solution was passed through a disposable syringe filter "CHROMAFIL" with a pore size of 0.45 μm, and then analyzed.

A freeze-dried chlorin e6 sample was used as a standard. As a result of the proposed processing of the reaction mixture, a practically non-degradable dosage form is obtained during storage. The decrease in the optical density of the solution over 2 years was only 0.1%, which is many times higher than the stability of all currently known drugs of the FS class.

Thus, the invention allows to obtain FS with high consumer properties, long shelf life, maximum purity.

Claims (2)

1. Photosensitizer for photodynamic therapy (PDT) in freeze-dried form, including chlorin e6 salt and meglumine as a cryostabilizer, characterized in that it contains trismeglumine chlorin e6 salt as e6 chlorine and additional dextran as a cryostabilizer in the following ratio of components in mass percent:
chlorine salt e6 1-27 meglumine 5-50 dextran rest 2. A method of producing a photosensitizer for PDT according to claim 1, comprising dissolving methylpheophorbide a in acetone, treating the resulting solution with aqueous alkali NaOH or KOH, followed by neutralizing the reaction mixture with dilute hydrochloric acid, separating the precipitated chlorine e6 precipitate, followed by washing, treating the precipitate with an aqueous solution and lyophilization, characterized in that the treatment of a solution of methylpheophorbide a in acetone with aqueous alkali is carried out at a temperature of 51-60 ° C, neutralization of the reaction mixture with dilute hydrochloric acid by t to pH 4.0–4.5, separation of the chlorine e6 precipitate is carried out by filtration through a celite 545 layer, washing of the chlorin e6 precipitate is carried out with deionized water, then chlorin e6 is extracted from celite with an aqueous solution of meglumine and dextran until the concentration of chlorin e6 salt in the solution is reached, the corresponding optical density D = 225-235 / 1 ml at a wavelength of maximum absorption of 661 nm and a solution pH of 9.30-9.35.