RU2576025C1 - Method for producing composition for photodynamic therapy in form of phospholipid nanoparticles based on glucamine chlorine e6 salt, maltose and phosphatidylcholine - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is described is a method for producing a pharmaceutical composition for photodynamic therapy in the form phospholipid nanoparticles based on chlorine E6 bis(N-methyl-D-glucamine)monosodium salt, maltose and phosphatidylcholine. The method consists in adding an aqueous solution of N-methyl-D-glucamine and a solution of sodium hydroxide to an aqueous suspension of chlorine E6. That is followed by adding an aqueous solution of maltose and mixing with an aqueous emulsion of phosphatidylcholine. Further, the produced suspension is exposed to 6-8 homogenisation cycles under pressure 600-1000 atm at temperature 40-50°C, which is followed by sterilisation filtration and lyophilisation.

EFFECT: invention provides implementing the above application.

2 cl, 3 tbl, 1 ex

Description

The invention relates to the field of pharmaceuticals and medicine and relates to a method for producing a pharmaceutical composition for photodynamic therapy in the form of phospholipid nanoparticles based on bis (N-methyl-D-glucamine) monosodium salt of chlorin E6, maltose and phosphatidylcholine.

One of the modern approaches in the development of new-generation drugs is the construction of original forms based on substances of a known spectrum of activity using modern technologies that make it possible to obtain drugs with high bioavailability, therapeutic efficacy and reduced side effects.

New technologies that can "extend the life" of known drugs include the development of drug transport systems in the body.

The most significant successes were achieved in the development of transport systems based on phospholipid nanoparticles, which have several advantages over others. They are non-toxic, biodegradable, do not cause allergic reactions, have a high affinity for cell membranes. One of the earliest systems of transport of biologically active substances in the body was liposomes.

Chlorin E6 is one of the most effective and successfully used in medical practice photosensitizers for the treatment of cancer and other neoplasms by the method of photodynamic therapy (FTD). Chlorin E6 has a number of spectral, physico-chemical and energy characteristics that distinguish it from other photosensitizers. For example:

- the presence of an absorption band in the long wavelength region of the visible spectrum (with a maximum of 662 nm in biological media), which corresponds to the region of greatest optical transparency for biological tissues;

- high contrast index;

- a high level of accumulation in target tissues in comparison with intact tissues;

- high photodynamic activity;

- relatively low phototoxicity;

- fast enough, within 24-48 hours, excretion from the body.

For the first time, water-soluble chlorophyll derivatives were used by E. Snyder (USA) in 1942, and were used for the prevention and treatment of cardiovascular diseases, atherosclerosis, and rheumatoid arthritis.

In the scientific literature on the use of derivatives of the chlorin series for photodynamic therapy was announced in 1986. In 1994-2001. in Russia A.V. Reshetnikov [1] developed a technology for extracting a complex of biologically active chlorins from plant materials, which contain chlorine E6 as the main component, the photocytotoxic effect of which on the tumor is enhanced, and the general pharmacological parameters are improved due to two other natural chlorins contained in the extract.

For two decades, intensive searches have been conducted for the most effective photosensitizers. Currently, around 20 different compounds for PDT have been developed and are undergoing clinical studies in the world.

Given that FTD has been included in the treatment standards since 2010, chlorin E6 and its derivatives are in demand on the domestic pharmaceutical market and are increasingly used, for example, for the treatment of cancer of the cervix, bladder, and various mucous membranes. However, despite the demand, chlorin E6 as a substance has several disadvantages that limit its widespread consumption, namely: the liquid form of finished drugs based on chlorin E6; short shelf life and loss of optical properties; elimination from the body within 36-48 hours or more; phototoxicity.

In this regard, an extremely urgent task is the search for new drugs for PDT and the development of effective dosage forms for existing drugs, in particular, based on E6 chlorin.

One of the most convenient, suitable for PDT dosage forms of chlorin E6, devoid of the above disadvantages, is a composition based on chlorin E6 included in phospholipid nanoparticles.

It is known that chlorin E6 is a hydrophobic substance, and its incorporation into phospholipid nanoparticles is a multi-stage process.

A known composition of the drug in the form of phospholipid nanoparticles for photodynamic therapy, including the drug, phosphatidylcholine and maltose and a method for its preparation (patent RU 2391966, 06/20/2010). Moreover, in the case of hydrophobic drugs, such as chlorin E6, the method of obtaining a composition in the form of phospholipid nanoparticles requires the use of additional solvents, such as ethanol, which complicates the process and requires additional purification from the solvent. Therefore, more often chlorin E6 is used in the form of its salts.

Known used as a photosensitizer (PS) drug Radachlorin (US Pat. RU 2183956, 06.27.2002), which is a mixture of sodium salts of several related chlorins, as well as other biologically active compounds, for example purpurin, chlorine p6. The methylpheophorbide method used to obtain Radachlorin does not allow the preparation of a preparation in the form of chlorin E6 with a high degree of purity.

The drug Photoditazine (RU 2144538, 01/20/1998) is an N-methyl-D-glucamine salt of chlorin E6 and is a 0.5% aqueous solution containing polyvinylpyrrolidone. Despite the fact that the preparation is almost pure chlorin E6 (the content of chlorine impurities is not more than 2.5%), its shelf life is small (less than 1 year), and special storage conditions at low temperatures are necessary.

The drug Photolon (RU 2152790, 07.20.2000), is a complex of trisodium salt of chlorin E6 with polyvinylpyrrolidone in the form of a lyophilisate for the preparation of a solution for intravenous infusion. The drug is stable during prolonged storage at room temperature, but also represents, in addition to pure chlorin E6, impurities of derivatives of chlorins and porphyrins.

The main disadvantage of the currently used PS for PDT based on chlorin E6 is their liquid dosage form, which is unstable during long-term storage and difficult to standardize due to the presence of various chlorins and their derivatives in it. In this regard, the most optimal dosage form for preparations based on chlorin E6 is lyophilized.

Patent RU 2367434, September 20, 2009 describes a photosensitizer for photodynamic therapy in freeze-dried form, which is a mixture of bis (N-methyl-D-glucamine) monosodium salt of chlorin E6 and maltose in a ratio of 1: 1, as well as a method for its preparation, which consists in in that chlorin E6 is suspended in water, N-methyl-D-glucamine and sodium hydroxide are added, the resulting solution of chlorin E6 salts is filtered, maltose is added and lyophilized. In this method, a filtration step is used to isolate salts, which also complicates the process.

The preparation of a pharmaceutical composition for photodynamic therapy in the form of phospholipid nanoparticles based on bis (N-methyl-D-glucamine) monosodium salt of chlorin E6, maltose and phosphatidylcholine is not described in the literature.

This developed drug for photodynamic therapy, in comparison with drugs on the market, has several advantages:

- high accumulation in the tumor;

- high contrast ratio;

- fast excretion from the body;

- the small size of the nanoparticles (up to 30 nm), which prevents the degradation of the drug by the reticuloendothelial system, and also improves its pharmacodynamics (biotransformation);

- lyophilized dosage form increases the shelf life of the drug, convenient to use and transportation.

Therefore, the development of a simple method for producing a highly effective pharmaceutical composition for photodynamic therapy in the form of phospholipid nanoparticles based on bis (M-methyl-D-glucamine) monosodium salt of chlorin E6, maltose and phosphatidylcholine is an urgent task.

In accordance with the invention, a method is described for preparing a pharmaceutical composition for photodynamic therapy in the form of phospholipid nanoparticles based on bis (N-methyl-D-glucamine) monosodium salt of chlorin E6, maltose and phosphatidylcholine, characterized in that an aqueous suspension of chlorin E6 is added with stirring a solution of N-methyl-D-glucamine and a solution of sodium hydroxide in a molar ratio of 1: 2: 1, followed by the addition of an aqueous solution of maltose (weight. relative. 1:40 based on chlorine E6). The resulting solution is mixed with an aqueous emulsion of phosphatidylcholine (weight. 1:10 based on chlorine E6), and the resulting emulsion is subjected to several cycles (6-8) of homogenization under high pressure of 600-1000 atm at a temperature of 40-50 ° C followed by neutralization with an aqueous solution of alkali. The resulting solution is collected in a separate container, filtered through a 3-5 micron prefilter, followed by sterilizing filtration through a 0.22 micron filter and lyophilization.

The method does not require the use of toxic solvents and stages for the isolation of individual salts of chlorin E6, since the chlorin salt obtained from the reaction of chlorin E6 with N-methyl-D-glucamine and sodium hydroxide is mixed without isolation with an aqueous solution of maltose and an aqueous emulsion of phosphatidylcholine (weight. calculated on chlorin E6: 1:40:10), the suspension obtained is subjected to several homogenization cycles under high pressure of 600-1000 atm at a temperature of 40-50 ° C, followed by sterilizing filtration and lyophilization.

The method is simple to implement and allows you to get a highly effective stable pharmaceutical composition for photodynamic therapy based on bis (N-methyl-D-glucamine) monosodium salt of chlorin E6, in the form of phospholipid nanoparticles up to 30 nm in size and having a high percentage of inclusion of chlorin E6 in phospholipid nanoparticles - up to 94%, which has several advantages over other compositions for photodynamic therapy. These advantages are: a high percentage of the inclusion of the glucamine salt of chlorin E6 in phospholipid nanoparticles, high therapeutic activity and storage stability. The solid dosage form of FS based on chlorin E6 allows you to keep the drug at room temperature for 3 years and is easily transported.

The implementation of the above method provides a means for PDT based on chlorin E6, incorporated into phospholipid nanoparticles up to 30 nm in size, having a high percentage of inclusion of chlorin E6 in nanoparticles - up to 94%, exhibiting high therapeutic activity, with a long shelf life (up to 3 years ) and not requiring special storage conditions.

Examples of carrying out the invention.

Inclusion of chlorin E6 salt in phospholipid nanoparticles.

Two solutions are prepared, water for injection is used.

Solution A. 30.0 g of Lipoid S 100 are dissolved in 200 ml of water (pH 6.7) with vigorous stirring.

Solution B. 3.0 g of chlorin is suspended in 100 ml, 300 ml of an aqueous solution of 1.98 g of N-methyl-D-glucamine are added, a solution of 0.2 g of NaOH in 50 ml of water, mix thoroughly, incubated for 30 minutes and add chilled. to room temperature, a solution of 120 g of maltose in 200 ml of water (maltose is dissolved by heating to 40 ° C).

Solutions A and B are mixed and thoroughly mixed with a mechanical stirrer for 40 minutes until a homogeneous emulsion is obtained.

The emulsion obtained is homogenized using a Mini-Lab 7.30 N high pressure homogenizer, Rannie, Denmark (APV homogenizer). After each cycle, sampling is carried out to determine the particle size.

The parameters of the homogenization process: pressure 600 - 1000 atm, suspension temperature - 45-50 ° C, homogenization time - 5-7 minutes, the number of process cycles 6-8. At the end of homogenization, the pH of the solution is monitored. If necessary, the pH of the resulting suspension is adjusted to 7.0-7.6 by titration with a 1N NaOH solution, then it is filtered successively through a 3-5 micron prefilter and a 0.22 micron filter, poured into vials and subjected to freeze-drying. The vials are closed with rubber stoppers, wrapped in aluminum caps and marked.

The resulting preparation is analyzed. Examples of the preparation and the results of its analytical control are shown in tables 1, 2.

Analytical parameters of chlorin E6 samples in the composition of phospholipid nanoparticles.

Table 1 N p / p Analytical indicators Requirements of the draft ND Test results Process parameters pressure during homogenization - 600 atm tem-pa suspensions 42 ° C; number of homogenization cycles -6 one. Particle size ≤50 nm 22.8-23.4 2. Vial content - chlorin E6 (HPLC) 47.5-52.5 mg 47.8-49.2 mg - phosphatidylcholine (UV spectrometry) 475-525 mg 479-493 mg 3. The inclusion of chlorin E6 in phospholipid nanoparticles ≥80% 89.2-92.3%

table 2 N p / p Analytical indicators Requirements of the draft ND Test results Process parameters pressure during homogenization - 1000 atm tem-pa suspensions 48 ° C: number of homogenization cycles - 8 one. Particle size ≤50 nm 21.3-22.4 2. Vial content - chlorin E6 (HPLC) 47.5-52.5 mg 48.7-49.8 mg - phosphptylcholip (UV spectrometry) 475-525 mg 482-496 mg 3. The inclusion of chlorin E6 in phospholipid nanoparticles ≥80% 92.2-94.0%

The optical properties of Chlorin-NF were studied during storage at room temperature in three series of the preparation.

To study the stability of the drug, the optical densities of the drug solutions were measured at two wavelengths and their ratio was calculated. The ratio of optical density at a wavelength of 403 nm to optical density at a wavelength of 655 nm should be at least 4.4. For this, 10 mg (accurately weighed) of the freeze-dried powder was dissolved in 200 ml of purified water. The optical density was measured. It was shown that the drug is stable for 3 years (table 3).

Table 3 The study of the stability of the drug K = A403 / A655 At the time of release 6 months 1 year 1 year 6 months 2 years 2 years 6 months 3 years 3 years 6 months Episode 1 7.9 7.8 7.6 7.4 7.2 7.3 7.1 6.9 Series 2 7.7 7.6 7.5 7.5 7.4 7.4 7.2 7.0 Series 3 7.8 7.6 7.3 7.3 7.4 7.2 7.3 7.0

Studies of the toxicity of the drug "Chlorin-NF" (production parameters are shown in table 2), intended for photodynamic therapy of malignant neoplasms, in two types of laboratory animals - mice and rats.

Acute toxicity:

LD ₅₀ for mice - 258 ± 29

LD ₅₀ for rats - 240 ± 16

A possible cause of death of mice from a single intravenous administration of the drug “Chlorin-NF” in lethal doses was acute cardiovascular failure.

The study of the "chronic" toxicity of the drug "Chlorin-NF" with repeated (within 30 days) intravenous administration in the range of the studied doses of 1, 12 and 40 mg / kg in rats and 25 and 50 mg / kg in dogs.

It was shown that the administration of the drug does not affect the appearance, general condition and behavior of animals, does not adversely affect the biochemical parameters of blood and the basic physiological functions of the body, does not cause pathomorphological changes, does not cause irritation, inflammation or tissue destruction at the injection site, which indicates about the good tolerance and harmlessness of the drug.

In in vivo experiments with mice inoculated with a tumor inoculated with Lewis lung carcinoma, the photoinduced antitumor activity of Chlorin-NF was studied.

For irradiation, we used an LED red light source developed at FSUE “SSC NIOPIK” with a maximum of 661 ± 16 nm. The diameter of the light spot was 1.0 cm; the distance from the LED to the irradiated surface was 2 mm. It was shown that when irradiated 15 minutes after intravenous administration of the drug at a dose of 5.0 mg / kg, a light source with an energy density of 90 J / cm ^{2 the} antitumor activity was:

tumor growth inhibition (SRW) 77.6-100%;

increase in life expectancy (VL) - 85.4%;

cure rate (CI) - 40%;

the drug is excreted from the body within 24 hours.

Thus, a method for producing a highly effective safe composition for use in photodynamic therapy based on the glucamine salt of chlorin E6 equipped with a phospholipid nanosystem of transport has been developed. The method is simple to implement and allows you to get a solid dosage form with an extended shelf life, convenient to use and transport.

Unlike the drugs available on the market, the developed drug has the following characteristics:

- high accumulation in tumor tissues;

- high contrast ratio;

- rapid elimination from the body (reduction of the phototoxic effect);

- the small size of the nanoparticles (up to 30 nm), which helps to protect the drug from degradation in the reticuloendothelial system, and also helps to improve its pharmacodynamics (absorption, bioavailability, metabolism);

- lyophilized dried form. This form increases the shelf life of the drug (up to 3 years), convenient in use and transportation.

Claims (2)

1. A method of obtaining a pharmaceutical composition for photodynamic therapy in the form of phospholipid nanoparticles based on bis (N-methyl-D-glucamine) monosodium salt of chlorin E6, maltose and phosphatidylcholine, characterized in that an aqueous solution of N- is added to the aqueous suspension of chlorin E6 with stirring methyl D-glucamine and a solution of sodium hydroxide in molar ratios of 1: 2: 1, followed by the addition of an aqueous solution of maltose at a weight ratio of chlorin E6 and maltose 1:40, the resulting solution is mixed with an aqueous emulsion of phosphatidylcholine at a weight ratio of chlorin E6 and phosphatidylcholine of 1:10 and the resulting emulsion is subjected to 6-8 cycles of homogenization under a pressure of 600-1000 atm at a temperature of 40-50 ° C, followed by sterilizing filtration and lyophilization. 2. The method according to claim 1, which includes the step of adjusting the pH of the resulting suspension to 7.0-7.6 with a NaOH solution before the sterilizing filtration step.