RU2367434C1 - Photosensitiser and method for preparing thereof - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: inventions cover preparing the biologically active compounds of chlorine series -photosensitiser (PS) for photodynamic therapy (PDT). The PS contains chlorine e6 with N-methyl-D-glucamine and sodium in molar ratios as follows 1:2:1, and a cryostabiliser. The PS has structural formula (I): Also, th invention discloses the method for making the photosensitiser for photodynamic therapy according to which chlorine e6 is suspended in apyrogenic water; N-methyl-D-glucamine and sodium hydroxide (NaOH) are added in stirring. The prepared chlorine e6 salt solution is filtered with the cryostabiliser added in estimated amount, and lyophilised. ^ EFFECT: said PS shows prolonged storage-stability, improved service properties and the consumer appeal, extended storage life as a medical preparation. ^ 4 cl, 1 ex, 1 tbl

Description

The invention relates to the field of pharmacology, in particular to the production of biologically active compounds of the chlorin series, in particular chlorine e6 derivatives, and can be used to produce chlorin e6 salts, which can be used as highly effective photosensitizers (hereinafter FS) for the photodynamic therapy of cancer and other neoplasms of various genesis, as well as fluorescence diagnostics of cancer cells.

Among the numerous derivatives of the chlorin series obtained from natural chlorophyll a and studied as PS, only chlorin e6 derivatives have found a real embodiment in medical practice and are widely used as approved drugs for PDT of cancer and other neoplasms (see the structural formula below)

Currently, several methods are known for producing such FSs, on the basis of which the technology for creating RADACHLORIN medical preparations (US Pat. RF No. 2183956 dated 06/27/2002 and US Pat. No. 6969765 of 11/29/2005), PHOTOLON (US Pat. RF No. 2152790 dated 20.07) was developed. .2000), PHOTODITAZINE (US Pat. RF No. 2144538 dated 01/20/1998), as well as N-aspartyl-chlorin e6 (US Pat Appl. No. 20080021210) (a preparation registered in Japan, NPe6 or synonyms Laserphyrin, Talaporfin, Talaporfin Sodium )

So, RADACHLORIN is a mixture of sodium salts of at least two to three related chlorins, among which chlorin e6 is about 80-90% of the total mass of PS, the rest is purpurin 5 and chlorin p6. The presence of chlorine impurities, in the opinion of the authors of the drug, contributes to its greater effectiveness as a FS compared to pure chlorine e6. The presence of chlorine impurities in the RADACHLORIN preparation is due to the very method of producing chlorin e6 from pheophorbide a, which, in principle, cannot ensure the formation of high purity chlorin e6.

Another PS - PHOTOLON is a complex of trisodium salt of chlorin e6 with polyvinylpyrrolidone (PVP), and the mutual content of chlorin e6 and PVP can vary within very wide limits. In addition to chlorin e6, numerous chlorine and porphyrin derivatives were found in the preparation, the formation of which was also determined by the method of isolating the initial intermediate of pheophytin a from spirulina with a basic substance content of not higher than 90-95%. The use of such pheophytin a as a feedstock for the synthesis of chlorin e6 from it inevitably leads to a final preparation containing, in addition to chlorin e6, also a number of other related chlorins. The invention, according to the authors of the patent, allows to increase the yield and purity of the product up to 90-95%. The product has stability during prolonged storage in a lyophilized state at room temperature and, as the authors of the drug suggest, a higher specific activity compared to pure chlorine e6 and its salts.

PHOTODITAZINE is a chlorine e6 bis-N-methyl-D-glucamine salt in the form of a sterile 0.5% solution, which is a 0.5% aqueous solution of chlorin e6 N-methyl-D-glucamine salt containing polyvinylpyrrolidone. This PS contains chlorine e6 of a high degree of purity (97% and higher) with a chlorine content of not more than 2-2.5%. However, this aqueous solution is not stable enough and has a limited shelf life (not more than 1 year) when stored in a refrigerator at 4-8 ° C.

And finally, TALAPORFIN, is a tetrasodium salt of N-aspartyl-chlorin e6. This preparation is obtained from chlorin e6 in two stages: 1) treatment of chlorin e6 with carbodiimide with the release of intermediate anhydride; 2) the interaction of chlorin e6 anhydride with an aqueous solution of sodium aspartate and the selection of the target product. The addition of an aspartic acid residue to the chlorin e6 molecule apparently makes some positive contribution to the biodistribution of FS in organs and tissues during PDT, but it is not so significant as to radically change the scope of this FS compared to other FS based on chlorin e6.

All known FSs based on chlorin e6 for intravenous use are either aqueous solutions (concentration ~ 35 mg of chlorin e6 in 10 ml of water, i.e. 0.35% solution for RADACHLORIN or 0.5% solution for PHOTODITAZINE followed by dilution to 100 ml), or FOTOLON, a prepared solution of a powder of 100 mg of trisodium salt of chlorin e6 together with polyvinylpyrrolidone in 100 ml of water. Thus, the concentration of PS calculated by the content of chlorin e6 in the injection solution does not exceed 0.3-1.0 mg / ml.

Special studies have shown that chlorin e6 has the most pronounced photosensitizing effect, and the accompanying impurities, which for the most part easily aggregate in aqueous media, have a lower quantum yield of singlet oxygen generation. 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 FOTOLON and RADACHLORIN preparations blur the clear line between the cancerous tumor and nearby healthy tissue, which significantly reduces the effectiveness of PDT when working with this drug, since noticeable photodestruction and healthy tissue occur. Thus, the presence of impurities even of chlorin nature significantly reduces the effectiveness of the drug as a FS.

Of all the preparations presented above, FOTOLON has maximum storage stability, but at the same time relatively low tropicity, the RADACHLORIN preparation has approximately the same consumer properties as photolon, and the PHOTODITAZIN preparation has low storage stability with high tropicity.

The most significant drawback of dosage forms of drugs based on the use of aqueous solutions is their relatively low stability during prolonged storage even at a temperature of 8-12 ° C due to the occurrence of radical processes leading to partial destruction and transformation of the tetrapyrrole macrocycle.

Therefore, the most promising for the preparation of drug formulations based on chlorins is, of course, the creation of freeze-dried forms.

By technical nature (in its properties), the closest to the proposed photosensitizer is the PHOTODITAZINE photosensitizer for photodynamic therapy, containing at least 98% chlorin e6, N: methyl-D-glucamine in a 1: 2 molar ratio and polyvinylpyrrolidone with a molecular weight of 9600 up to 11500, while chlorin e6 has a certain structural formula (see US Pat. RU No. 2276976, class A61K 31/409, dated 10.08.2004).

However, this photosensitizer (PHOTODITAZINE) is not stable enough and has a limited shelf life.

By technical essence, the closest to the proposed method is a method for producing a photosensitizer for PDT, which consists in treating spirulina with an inorganic acid, stirring, filtering, followed by derivation of pheophorbide a derivatives, dissolving them in acetone, adding an inorganic base in the form of an aqueous solution by heating, cooling, neutralization with hydrochloric acid, centrifugation, separation of the precipitate containing the target product, and washing it with distilled water, while by centrifugation, the precipitate containing chlorin e6, after washing with distilled water, is suspended under sterile conditions in pyrogen-free water, re-centrifuged, the precipitate obtained is dissolved in an aqueous solution of N-methyl-D-glucamine, the pH is adjusted to a certain value, the required amount of pyrogen-free water is added, the solution is filtered and add polyvinylpyrrolidone (see US Pat. RU No. 2276976, class A61K 31/409, from 10.08.2004).

However, the use of this method allows to obtain only an aqueous solution of FS, which is not stable enough and has a limited shelf life.

The technical result of the proposed inventions is the development of PS, more stable during prolonged storage even at room temperature, with improved performance and consumer qualities, increased shelf life of it as a medical product and simplification of its practical use in medical institutions, as well as the development of a method for its production (manufacture ), allowing to obtain a FS having the above operational and consumer properties.

This is achieved by the fact that the photosensitizer for photodynamic therapy in freeze-dried form, according to the invention, contains a bis (N-methyl-D-glucamine) monosodium salt of chlorin e6 of the formula (1)

and a cryostabilizer in a ratio of 1: 1 weight parts.

In addition, maltose is taken as a cryostabilizer.

According to the second technical solution, chlorin e6 is taken in a method for producing a photosensitizer for photodynamic therapy and suspended in pyrogen-free water, the calculated amount of N-methyl-D-glucamine and sodium hydroxide (Na OH) is added with stirring, the resulting solution of chlorine e6 salts is filtered, added to him the calculated amount of cryostabilizer and lyophilize. In addition, chlorin e6, N-methyl-D-glucamine and sodium hydroxide are taken in the following molar ratios: 1: 2: 1, while the cryostabilizer is taken in a weight ratio of 1: 1 to the salts of chlorin e6, and maltose is taken as a cryostabilizer.

A comparison of the proposed technical solutions with the known ones allows us to confirm compliance with the criterion of "novelty", and the absence of distinctive features in the analogues indicates the compliance with the criterion of "inventive step".

Preliminary tests suggest the possibility of wide industrial use.

Essentially, a new method for producing FS based on chlorin e6 is proposed. This method is characterized in that a composition of chlorin e6 is used to produce PS, which is obtained by chemical modification of methylpheophorbide a, followed by the addition of N-methyl-D-glucamine (C ₇ H ₁₇ NO ₅ ), sodium hydroxide and a cryostabilizer (maltose) taken in certain proportions, which is a freeze-dried powder, stable when stored at room temperature. This method is implemented by first obtaining methylpheophorbide and a high degree of purity and then obtaining a photosensitizer by the following methods.

The chlorin e6 salt has the following structural formula

Benefits:

1. Lyophilization of the chlorin e6 salt of formula (1) improves the stability of the FS during storage and facilitates the lyophilization process.

2. The addition of a cryostabilizer improves the lyophilization conditions (it increases the porosity of the resulting tablet, significantly accelerates the lyophilization process, reduces the dissolution time during the preparation of the injection solution, and significantly increases the stability of the drug even when stored at room temperature).

3. Lyophilization of FS can significantly increase the preservation of the medicinal product and leforms prepared from it.

4. The use of powdered FS greatly simplifies the preparation of a variety of formulations of ointments, creams and gels used for PDT of surface forms of cancer and other diseases in ophthalmology, otolorology, dentistry, gynecology, etc.

Example. To a solution of 4 g of methylpheophorbide a in 700 ml of acetone at 40-45 ° С add 150 ml of a 10% solution of potassium hydroxide (KOH) with intensive argon sparging, after 0.5 hours the temperature of the solution is brought to 60 ° С, 650 ml of 10% are added KOH solution is kept for 1 h under argon, controlling the conversion of methylpheophorbide a to chlorin e6 by HPLC (high pressure liquid chromatography) and, after completion of the process, the solution is cooled to 15-20 ° С, the reaction mixture is neutralized with dilute hydrochloric acid to pH 4.5- 5, the precipitate is separated cent by washing, washed 5 times with pyrogen-free distilled water, followed by re-centrifugation, then, under sterile conditions, the precipitate is suspended in pyrogen-free water, 0.5 L of a solution of N-methyl-D-glucamine and NaOH (calculated amount) is added to the suspension, then 3.5 g of maltose and the resulting solution is filtered through a 0.22 µm filter, lyophilize in 5 ml vials. Receive FS in the form of a porous tablet containing in a bottle of 35 mg of the main substance (in terms of chlorin e6).

Thus, the proposed technical solutions achieve the technical result.

A storage table confirming the improved properties of the claimed FS relative to the prototype.

Determination of storage stability of the photosensitizer (I) Stock solution In 3 months In 6 months After 9 months After 1 year D solution 0.455 D = 0.450 D = 0.450 D = 0.445 D = 0.445 one hundred% 99% 99% 97.8% 97.8%

Storage conditions - in the dark, in the refrigerator at + 6- + 10 ° С. Determination of the percentage of photosensitizer. Dissolve a sample of 10 mg of dry photosensitizer in 400 ml of distilled water, adjust the pH of the solution to 9.0 and determine the optical density of the solution at 655 nm. In this case, the density of the solution D = 0.455, calculated on the basis of the molar extinction coefficient for pure chlorin e6 (mol. Weight 596.68) = 36750 for the band at 655 nm, is taken as 100% (see US Pat. RU 2330037). The inventive photosensitizer contains bisglucumin sodium salt (mol. Weight 1009.1) with the same coefficient. pier extinction and maltose in a weight ratio of 1: 1, therefore 2.96 mg of chlorin e6 is contained in 10 mg of dry powder.

The claimed photosensitizer is not inferior to the prototype in biological and pharmacological activity.

Claims (4)

1. Photosensitizer for photodynamic therapy in freeze-dried form, characterized in that it contains a bis (N-methyl-D-glucamine) monosodium salt of chlorin e6 of the formula (I)

and cryostabilizer in a ratio of 1: 1 weight. h 2. The photosensitizer according to claim 1, characterized in that maltose is taken as a cryostabilizer. 3. The method of producing a photosensitizer for photodynamic therapy according to claim 1 or 2, characterized in that chlorine e6 is taken and suspended in pyrogen-free water, N-methyl-D-glucamine and sodium hydroxide (NaOH) are added with stirring in the following molar ratios: chlorin e6, N-methyl-D-glucamine and sodium hydroxide - 1: 2: 1, respectively, the resulting solution of chlorin e6 salts is filtered, a cryostabilizer is added to it in the ratio 1: 1 to the chlorin e6 salt by weight and lyophilized. 4. The method according to claim 3, characterized in that maltose is taken as a cryostabilizer.