RU2523380C1 - Photosensitiser and method of its obtaining - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention represents a photosensitiser (PS) for photodynamic therapy (PDT) of cancer and other neoplasms of different genesis, as well as fluorescent diagnostics, where PS contains a trismeglumin salt of chlorine e6, and as a cryostabiliser - methyl glucamine in a ratio of 1:0.1-0.2 wt.p. The invention also relates to a method of obtaining the photosensitiser for PDT, which includes dilution of methylpheophorbide a in acetone, processing the obtained solution with aqueous alkali (NaOH or KOH) with following neutralisation of a reaction mixture by diluted hydrochloric acid to pH 4.5-5.0, separation of the precipitated sediment of chlorine e6 with following washing with distilled water, processing the sediment with a water solution and lyophilisation, which is characterised by the fact, that processing of methylpheophorbide a in acetone with aqueous alkali is carried out at a temperature of 40-50°C, separation of chlorine e6 sediment is realised by filtering through a layer of celite 545, and chlorine e6 is extracted from celite by a water solution of meglumine until the concentration of chlorine e6 salt, corresponding to optic density D=225-235/1 ml, is achieved.

EFFECT: obtaining a lyophilised drug form PS without ballast substances, which has an increased storage stability, which results in simplification and optimisation of chlorine e6 solution with a high degree of purity.

2 cl, 2 ex, 2 dwg

Description

The invention relates to the field of pharmacology, namely 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 PSs for PDT for cancer and other diseases use derivatives of chlorin e6. 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. А61К 31/409] (Belmedpreparaty JSC is produced) 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 06/27/2002., US Pat 7550587 dated 06/23/2009] manufactured 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] (000 BETA-GRAND is produced) - 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.

The practical use of PS like PHOTOLON, RADACHLORIN or PHOTODITAZINE for intravenous (iv) use has some limitations due to the presence in their composition of a large number of by-products (PVP or related chlorins) with much lower solubility in water than chlorin e6 in the form meglumine salt, and therefore this process is usually carried out in the form of dilute solutions by the drip method. For this purpose, one vial of PS containing approximately 35 mg of PHOTODITAZINE or RADACHLORIN, or 100 mg of the trisodium salt of chlorin e6 in PHOTOLONE, is diluted to 100 ml of the solution, and ived for 30-40 minutes. It should be noted that the presence of PVP in the composition of the PS leformform imposes restrictions on the use of more concentrated solutions due to the increased viscosity of injection solutions and for the application of the iv injection method.

Also known is FS based on chlorin e6, which is a bismeglumine monosodium salt of chlorin e6, in the form of a lyophilized form in the presence of maltose as a cryostabilizer [see RU patent No. 2367434 CL, А61К 31/409, dated 09/20/20091. However, this FS is not used in practical medicine.

In addition, from the data (see www.drugs.com) the new FS-TALAPORFIN is known - mono-aspartyl-Chlorin e6 (Synonyms: Talaporfin Sodium, NPe6 or Laserphyrin), (CAS registry number 0110230-98-3), manufactured by the company Meiji Seika Kaishi (Japan), which is chlorin e6 amide with aspartic acid in the form of the tetrasodium salt (see US Pat. Appl. No. 20110105745. Publication date: 2011-05-05). Its preparation is based on the interaction of chlorin e6 with carbodiimide with the formation of previously unknown cyclic anhydride, which is then disclosed by the disodium salt of aspartic acid to Talaporfin. However, the cost of this drug is very high and amounts to 387208 yen (~ $ 4000) per bottle (100 mg).

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, 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 the 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 chlorin e6 is to obtain the initial chlorin e6 of the highest possible purity with the lowest possible technological problems.

Currently, there are several ways to obtain high-purity chlorin e6. One of them is the specific processing of “living” chlorella (Chlorella ellipsoidea) through the sequential isolation of pheophytin and its alkaline conversion to 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 chlorin 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) multiple 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 synthesis of chlorin e6 specially purified by chromatography from chlorin e6 trimethyl ether in low yield (about 25%) was recently carried out in the laboratory of K.M. Smith [Jinadasa, R. G, W., Ni, X., Vicente, MGH, and Smith, K. M. (2011) Syntheses and cellular investigations of 17 ³ -, 15 ² -, and 13 ¹ -amino acid derivatives of chlorin e6. J. Med. Chem., 54, 7464-7476].

However, this method is suitable only for the preparation of exclusively analytical amounts of chlorin e6, and, of course, cannot be used to obtain practically significant production quantities of PS for clinical studies. The production of chlorine e6 of high purity from methylpheophorbide and by its alkaline hydrolysis and a series of sequential chemical operations is also proposed in the patent [RU 2330037 C1 of 07.27.2008].

Since only chlorine e6 of a high degree of purity has the most pronounced photodynamic properties for a particular tumor or organ, the presence of chlorine impurities in the photosensitizer, with approximately the same photophysical and photodynamic properties in vitro experiments, but significantly different in their effects in vivo, significantly affects the consumer properties of the photosensitizer as a drug. Therefore, the main requirements for the creation of PS are the maximum possible quality (i.e., the purity of the initial substance), the stability of the dosage form and, if possible, the simplicity and reproducibility of the technological process.

All these requirements are implemented in our proposed method for producing a photosensitizer based on highly purified chlorin e6 from methylpheophorbide a.

By technical essence, the closest to the proposed drug is a PDT photosensitizer in freeze-dried form containing bis (N-methyl-D-glucamine) monosodium salt of chlorin e6 and cryostabilizer maltose in a ratio of 1: 1 weight.h. [cm. RU patent No. 2367434 C1, according to Cl. A61K 31/409, dated 09.20.20091].

However, this PS contains a large amount of ballast (maltose), which makes it difficult to obtain highly concentrated injection solutions for the implementation of the jet method in / in the introduction of the drug.

By technical nature, the closest to the proposed method is a method for producing FS for PDT, which consists in the fact that chlorin e6 is suspended in pyrogen-free water, N-methyl-D-glucamine (meglumine) and NaOH are added with stirring in the following molar ratios: chlorin e6, N-methyl-D-glucamine (meglumine) and NaOH - 1: 2: 1, respectively, the resulting solution was filtered, cryostabilizer maltose was added to it in the ratio 1: 1 to the chlorine salt e6 by weight and lyophilized [see RU patent No. 2367434 CL A61K 31 / 409J from 09/20/2009].

According to the above example, this method is implemented by preliminary obtaining chlorin e6 from methylpheophorbide a.

However, the use of a significant amount of maltose in this method does not allow to obtain highly concentrated injection solutions of PS, which complicates the implementation of the i.v. inkjet method and reduces the consumer properties of the drug.

The technical result of the proposed inventions is the development of FS, more stable during prolonged storage even at room temperature, with improved consumer qualities, simplifying its practical use in medical institutions, as well as developing a method for its preparation, which allows to obtain FS with high operational and consumer properties.

This is achieved by the fact that the PS for PDT in the lyophilized form, according to the invention, contains meglumine of the formula (I) as the chlorin e6 salt, and meglumine as a cryostabilizer in a ratio of 1: 0.1-0.2 parts by weight, in addition, an additional the amount of meglumine in excess of equimolar, i.e. three moles per 1 mol of chlorin e6, in an amount of 0.1-0.2 parts by weight it is necessary to bring pH = 9.30-9.35 in the solution, which creates the conditions for the complete ionization of all three carboxyl groups in the e6 chlorin molecule and, accordingly, to obtain a stable, water-soluble lexform, in addition, in the method for producing PS for PDT, including dissolving methylpheophorbide a in acetone, treatment of the resulting solution with aqueous alkali, followed by neutralization of the reaction mixture with dilute hydrochloric acid to pH = 4.5-5.0, separation of the precipitated chlorine e6 precipitate, followed by washing with distilled water, treated the precipitate with an aqueous solution of meglumine and lyophilization, according to the invention, the solution of methylpheophorbide a in acetone is treated with aqueous alkali only at 40-50 ° C, the precipitate of chlorin e6 is separated by filtration through a layer of celite 545 and chlorin e6 is recovered from celite 545 with an aqueous solution of meglumine to when the concentration of chlorin e6 salt corresponding to the optical density of 230-240 / 1 ml was reached in the solution at a wavelength of the absorption maximum of 655 nm and the pH of the solution was 9.30-9.35, the solution was filtered through 0.22 mmc millipores and lyophilized.

A comparison of the proposed solutions with the known ones makes it possible to claim compliance with the “novelty” criterion, and the absence of distinctive features in the analogues indicates compliance with the “inventive step” criterion. Preliminary tests allow us to judge the possibility of wide practical use.

The claimed photosensitizer is not inferior to the prototype in biological and pharmacological activity, and the lyophilized leform form created on its basis, containing 37.5-40 mg of the active substance in one bottle - chlorin e6, is optimal compared to other officially approved drugs for clinical use (PHOTOLON. RADACHLORIN, PHOTODITAZINE) and is stable for 2 years, as evidenced by the HPLC data of the stability of the leform under storage at a temperature of 8-12 ° C.

Thus, the constancy of the feedstock is due to the use of methylpheophorbide a, and the process of the chemical conversion of methylpheophorbide a to chlorine e6 is maximally unified and possible without loss of quality when using significantly larger loads. At the same time, the sterile aqueous solution obtained as a result of processing at the rate of 1 mol of chlorin e6 per three moles of meglumine allows one to obtain after pouring this solution of 10 ml into dark glass bottles and lyophilization, a photosensitizer called HELIOCHLORIN, which in terms of consumer qualities is stable at long-term storage (at least 2-3 years) and the amount of e6 chlorin, per 1 bottle, significantly exceeds FS PHOTODITAZINE and FS RADACHLORIN. The analysis of the obtained photosensitizer was carried out by high performance liquid chromatography (HPLC) (See Example No. 1). 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 = 4.5-5.0, separating the precipitated chlorine e6 precipitate, followed by washing with distilled water, treating the precipitate with an aqueous meglumine solution and lyophilization. According to the invention, the treatment of a solution of methylpheophorbide a in acetone with an aqueous alkali is carried out only at 40-50 ° 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 meglumine solution until the concentration of chlorin e6 salt corresponding to optical density of 230-240 1 ml at a wavelength of maximum absorption of 655 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 No. 1).

Thus, as a result of the proposed processing of the reaction mixture, a lexform practically non-degradable during storage was obtained. The decrease in the optical density of the solution over 2 years was only 0.29%, which is many times higher than the stability of all currently known drugs of the FS class.

EXAMPLE No. 1

To a solution of 4 g of methylpheophorbide a in 800 ml of acetone, 500 ml of 10% sodium hydroxide solution was added during 1 hour with intensive argon sparging, stirred for 2 hours at the same temperature, 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 the minimum amount of water and filtered through a layer of celite 545 with a height of 5 cm on a glass filter with a diameter of 8 cm, rinse the celite with distilled water until there are no inorganic salts in the filtrate, the upper layer of celite 545 containing chlorin e6 is separated, placed in a flask, add 150 ml of 1% solution of meglumine in pyrogen-free water, stirred for 5 minutes, the chlorin e6 solution is filtered off, another 250 ml of a 1% solution of meglumine is passed through celite, celite is additionally washed with another 600 ml of pyrogen-free water, the solution is filtered twice through Stericap filters are 0.22 mmol millipores, the total solution volume is adjusted to 1 l, if necessary, the solution pH is adjusted to 9.25-9.30, poured into 10 ml dark glass bottles, lyophilized under standard conditions and 100 photosensitizer vials are obtained in the form of porous tablets containing each bottle contains 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 bottle 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 No. 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 ° С;

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 chlorination of 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 obtained virtually non-degradable during storage of the leform. The decrease in the optical density of the solution over 2 years was only 0.29%, which is many times higher than the stability of all currently known drugs of the FS class.

Thus, the proposed technical solutions achieve the technical result:

increasing the stability of the lyophilized lexform FS during storage by optimizing the ratio of chlorin e6 salt and cryostabilizer; The absence of ballast substances in the leform of FS;

simplification and optimization of obtaining a solution of chlorin e6 of high purity, based on methylpheophorbide a.

Claims (2)

1. Photosensitizer for PDT in freeze-dried form, including chlorin e6 salt and cryostabilizer, characterized in that it contains trismeglumine chlorin e6 salt as e6 chlorine, and meglumine as cryostabilizer in a ratio of 1: 0.1-0.2 parts by weight. 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 to pH 4.5-5.0, separating the precipitated chlorine e6 precipitate, followed by washing with distilled water, treating the precipitate with an aqueous solution and lyophilization, characterized in that 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 filtration through a layer of celite 545, and chlorin e6 is removed from celite with an aqueous meglumine solution until the concentration of chlorin e6 salt in the solution reaches the corresponding optical density D = 225-235 / 1 ml at a wavelength of absorption maximum of 655 nm and a solution pH of 9.30-9.35.

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