Photosensitizer compositions and methods of making the same

Abstract

The present application relates to the field of pharmacy. Specifically, the present application discloses a photosensitizer composition and a preparation method thereof. The photosensitizer composition comprises chlorin e6 or a pharmaceutically acceptable salt thereof and a stabilizer, and the stabilizer comprises meglumine and low molecular weight (600-50,000 Da) chitosan. The photosensitizer composition of the present application has long-lasting stability and can be used for photodynamic therapy of cancer and other diseases.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field. Specifically, it relates to a photosensitizer composition and its preparation method. The method includes preparing dihydroporphyrin E6 triglucamine salt as a highly efficient photosensitizer in a lyophilized state. This photosensitizer can be used for photodynamic therapy of cancer and tumors caused by various reasons, as well as for fluorescence diagnosis of cancer cells, etc. Background Technology

[0002] Photosensitizers, such as dihydroporphyrin E6 derivatives, have been approved for photodynamic therapy of cancer and other diseases and are widely used in medicine.

[0003] Photosensitizer Photolon (manufactured by "Bell Medical Preparations" JSC) is listed in the Russian Drug Catalogue (…). www.rlsnet.ru The substance is a freeze-dried powder of trisodium dihydroporphyrin E6 mixed with some polyvinylpyrrolidone (see Russian Patent Application Publication No. 2152790, Publication Date: July 20, 2000, HISakau, TV Trukhacheva, AL Zhebentyaev, PTP Petrov. HPLC study of chlorine E6 and its molecular complex with polyvinylpyrrolidone. Biomedical Chromatogr., (2007), 21, 318-325; HISakau, TV Trukhacheva, PTP Petrov. Isolation and identification of impurities in chlorine E6. J. Pharmaceutical and Biomedical Analysis, (2007), 45, 20-29).

[0004] The photosensitizer Radachlorin (manufactured by Rada-Falma Ltd.) is an aqueous solution composed of a mixture of several indeterminate dihydroporphyrins, the main component of which is trisodium dihydroporphyrin E6 (accounting for 80-90% of the total mixture containing polyvinylpyrrolidone).

[0005]

[0006] (See Russian Patent Application Publication No. 2183956, Publication Date: June 27, 2002; and US Patent No. 7550587B2, Grant Date: June 23, 2009).

[0007] The photosensitizer FOTODITAZIN (manufactured by “Veta-Grand” GmbH) is an aqueous solution consisting mainly of a mixture of di-N-methyl-D-glucosamine dihydroporphyrin e6 and a small amount of dihydroporphyrin mixed with polyvinylpyrrolidone (not exceeding 2-4%) (see Russian Patent Application Publication No. 2276976, publication date: May 27, 2006).

[0008] Photosensitizers of the Photolon, Radachlorin, or FOTODINAZIN type are permitted for clinical use. However, these types of photosensitizers cannot be used for intravenous injection because they contain high levels of polyvinylpyrrolidone, which significantly increases the viscosity of the injection solution. Therefore, these drugs can only be used as diluents via intravenous infusion. A small vial of the above preparation (35 mg) is diluted to 100 ml of solution and infused intravenously over 30-40 minutes. Furthermore, the dihydroporphyrin E6 in these preparations differs from other dihydroporphyrins because the dihydroporphyrin E6 (trisodium salt and diglucamine salt) readily polymerizes in aqueous media and has a low quantum yield of singlet oxygen oscillations. This can lead to the erosion and disruption of the clear boundary between cancer cells and nearby healthy tissue, reducing the efficiency of photodynamic therapy and causing significant damage to healthy tissue.

[0009] The photosensitizer talaporfin (dihydroporphyrin e6 monoaspartic amide) (also known as Talaporfin Sodium, NPe6, Laserphyrin, CAS Registry No. 0110230-98-3), manufactured by Meiji Seika Kaishi in Japan, is a tetrasodium salt form of dihydroporphyrin e6, an "aspartic amide" (see US Patent Application No. 20110105745A1, publication date: May 5, 2011). Its preparation method involves the interaction of dihydroporphyrin e6 with carbodiimide to form a cyclic dehydrating compound.

[0010]

[0011] The ring structure then opens to form the disodium salt of aspartic acid, namely tarapofine:

[0012]

[0013] The main drawback of this drug is its high cost (JPY387,208 (~US$4,000) / 100mg).

[0014] Existing photosensitizers used in photodynamic therapy are lyophilized, with a weight ratio of dihydroporphyrin E6 triglucamine salt and the stabilizer meglumine in the range of 1:0.1–0.2 (see Russian Patent Application Publication No. 0002523380, publication date: July 20, 2014). However, this photosensitizer accumulates in damaged and tumor tissues for extended periods, prolonging patient recovery time, and cannot effectively break down damaged and tumor tissues. Furthermore, the meglumine in the photosensitizer cannot guarantee sufficient stability.

[0015] Existing methods for manufacturing high-purity dihydroporphyrin E6 include obtaining trisodium dihydroporphyrin E6 by thoroughly precipitating trisodium chlorophyll and its alkaline products from specially treated "active" Chlorella vulgaris (Elliptic Chlorella).

[0016]

[0017] Its main substance content reaches 93-98%, and the final yield is no more than 1% (see US Patent No. 8,349,335B2, grant announcement date: January 8, 2013). The main method for preparing dihydroporphyrin E6 according to this patent includes the following essential steps: 1) repeatedly washing the "active" Chlorella with inorganic salts; 2) gradually washing the "Chlorella" with an ethanol aqueous solution to remove polar impurities; 3) extracting chlorophyll with anhydrous pure ethanol; 4) treating the chlorophyll extract obtained by ethanol extraction with 1N hydrochloric acid to pH 2.5, and separating and filtering the pheophytin precipitate; 5) purifying the pheophytin using neutral alumina gradient chromatography in a system of hexane and dichloromethane; 6) dissolving the crystallized pheophytin in acetone, adding 1N NaOH, and adjusting the pH to 12; allowing the alkaline solution to stand for 12 hours, and filtering to precipitate trisodium dihydroporphyrin E6 crystals; 7) dissolving the dihydroporphyrin E6 salt in water, filtering the insoluble matter, and drying the aqueous solution.

[0018] Existing methods also include: specifically purifying "trimethyl ether dihydroporphyrin e6" using dihydroporphyrin chromatography, although the purification rate is very low (not exceeding 25%) (see Jinadasa RGW, Hu X., Vicente MGH, Smith KM, 17 dihydroporphyrin e6). 3 -,15 2 - and 13 1 - Synthesis and Cellular Studies of Amino Acid Derivatives (J. Med. Chem. 2011, 54, 7464-7476). This method is only applicable to the analysis of data (quantities) of dihydroporphyrin e6 and cannot be used for the preparation of large quantities of photosensitizers for clinical studies.

[0019] Furthermore, a method for preparing photosensitizers for photodynamic therapy involves simultaneously adding and stirring dihydroporphyrin E6, N-methyl-D-glucosamine (meglumine), and NaOH suspended in pyrogen-free water at a ratio of 1:2:1. The solution is then filtered, and maltose, a stabilizer, is added to the dihydroporphyrin E6 salt at a weight ratio of 1:1, followed by lyophilization (see Russian Patent Application Publication No. 02367434, publication date: September 20, 2009). However, this method uses a large amount of maltose as a stabilizer, making it unsuitable for preparing high-concentration photosensitizer injection solutions, which is difficult for intravenous administration and thus reduces consumer demand for this drug.

[0020] The closest prior art to this invention is Russian Patent Application Publication No. 0002568597 (publication date: November 20, 2015), which discloses a photosensitizer and its preparation method. The photosensitizer for photodynamic therapy, in a freeze-dried state, comprises a dihydroporphyrin E6 salt and a stabilizer. The dihydroporphyrin E6 salt includes triglucamine dihydroporphyrin E6, and the stabilizer includes meglumine and other dextran, and is composed of the following weight percentages: dihydroporphyrin E6 salt 1-27, meglumine 5-50, and dextran 23-94. The preparation method includes: dissolving methyl pheophylate a in acetone, treating the resulting solution with an aqueous solution of NaOH or KOH, neutralizing the reaction mixture with dilute hydrochloric acid, separating the precipitate of dihydroporphyrin E6, washing, rinsing with an aqueous solution, and then freeze-drying. Specifically, methyl methacrylate (MDMA), soluble in acetone, was treated with an alkaline aqueous solution at 51-60℃. The reaction mixture was neutralized with dilute hydrochloric acid to control the pH at 4-4.5. The precipitate of dihydroporphyrin E6 was separated and filtered through a layer of diatomaceous earth 545. The precipitate was then washed with deionized water. Dihydroporphyrin E6 was extracted from the diatomaceous earth using a solution of meglumine and dextran to obtain a concentrated solution of dihydroporphyrin E6 salt. Under conditions of a maximum absorption wavelength of 661 nm and a solution pH of 9.30-9.35, the corresponding optical density (OD) was 225-235 / mL.

[0021] The drawbacks of existing photosensitizers are that their components, meglumine and dextran, cannot guarantee complete stability. Large amounts of stabilizer (dextran) are difficult to prepare into highly concentrated injectable solutions for intravenous injection. Photosensitizers accumulate in damaged and tumor tissues for extended periods, prolonging the patient's recovery time, and they cannot effectively break down damaged and tumor tissues.

[0022] Existing methods for preparing photosensitizers have drawbacks: Treatment of methyl pheophylate a in acetone with an alkaline aqueous solution at 51-60℃ leads to partial evaporation of acetone and incomplete hydrolysis of methyl pheophylate a, resulting in impurities. Furthermore, oxidation of the reaction mixture occurs at 60℃, forming additional impurities. The structure of methyl pheophylate a is as follows:

[0023]

[0024] To address the aforementioned shortcomings, there is still a need in the art to improve photosensitizer compositions and their preparation methods. Invention Overview

[0025] The purpose of this invention is to provide a novel photosensitizer composition that can be stored for a longer period of time, simplifying its application in healthcare institutions and thus effectively expanding the consumer base.

[0026] The photosensitizer composition of the present invention can be used in photodynamic therapy and is stored in freeze-dried form, comprising dihydroporphyrin E6 or a pharmaceutically acceptable salt thereof and a stabilizer. The pharmaceutically acceptable salt of dihydroporphyrin E6 comprises dihydroporphyrin E6 triglucamine salt, and the stabilizer is meglumine and low molecular weight (600-50,000 Da) chitosan, combined in the following weight percentages: dihydroporphyrin E6 salt 1-1.1; meglumine 0.4-0.5; low molecular weight chitosan 0.09-0.11. The structure of dihydroporphyrin E6 triglucamine salt is as follows:

[0027]

[0028] C 55 H 87 N7O 21

[0029] MW1182.33

[0030] The method for preparing the photosensitizer composition of the present invention includes dissolving methyl methacrylate α in acetone, treating the solution obtained in the previous step with an aqueous solution of NaOH or KOH, then neutralizing the reaction mixture with dilute hydrochloric acid, separating the dihydroporphyrin E6 precipitate, washing the precipitate, rinsing it with an aqueous solution, and then freeze-drying it. According to the present invention, preferably, in an ultrasonic bath at 38-42°C (frequency 20-80 kHz), methyl methacrylate dissolved in acetone is treated with an alkaline aqueous solution, and then neutralized with dilute hydrochloric acid until the pH reaches 4-4.5. The precipitate of dihydroporphyrin E6 is separated, filtered through a layer of diatomaceous earth 545, and then washed with deionized water. Dihydroporphyrin E6 is then extracted from the diatomaceous earth with meglumine and a low molecular weight (600-50,000) chitosan aqueous solution to obtain a concentrated solution of dihydroporphyrin E6 salt. Under the solution environment of a maximum absorption wavelength of 661 nm and pH 9.30-9.35, the corresponding optical density (OD) is 225-235 / mL.

[0031] Specifically, the present invention relates to a photosensitizer composition comprising dihydroporphyrin E6 or a pharmaceutically acceptable salt thereof and a stabilizer, wherein the stabilizer comprises meglumine and low molecular weight chitosan.

[0032] According to the present invention, in the above photosensitizer composition, the contents of each component by weight percentage are 1-1.1% of dihydroporphyrin E6 or its pharmaceutically acceptable salt, 0.4-0.5% of meglumine, and 0.09-0.11% of low molecular weight chitosan.

[0033] According to the present invention, the low molecular weight chitosan is 600-50,000 Da, for example 1 KDa, 2 KDa, 3 KDa, 4 KDa, 5 KDa, 6 KDa, 7 KDa, 8 KDa, 9 KDa, 10 KDa, 15 KDa, 20 KDa, 25 KDa, 30 KDa, 35 KDa, 40 KDa, etc.

[0034] According to the present invention, the pharmaceutically acceptable salt of dihydroporphyrin E6 is dihydroporphyrin E6 triglucamine salt.

[0035] According to the present invention, the low molecular weight chitosan is deacetylated chitosan.

[0036] The photosensitizer composition of the present invention is preferably a lyophilized formulation.

[0037] The present invention also relates to a method for preparing the above-mentioned photosensitizer composition, comprising the following steps:

[0038] (1) The organic solvent solution of methyl pheophylate a was treated with an alkaline aqueous solution in an ultrasonic bath at 38-42℃.

[0039] (2) Neutralize with dilute acid.

[0040] (3) The precipitate of dihydroporphyrin E6 was separated by diatomaceous earth filtration.

[0041] (4) Dihydroporphyrin E6 was extracted from diatomaceous earth by adding meglumine and a low molecular weight chitosan aqueous solution, and

[0042] (5)Optionally freeze-dry the extracted dihydroporphyrin E6.

[0043] According to the present invention, the alkaline aqueous solution is selected from NaOH or KOH aqueous solution.

[0044] According to the present invention, the frequency of the ultrasonic wave is set to 20-80 kHz.

[0045] According to the present invention, the organic solvent solution of methyl pheophylate a is an acetone solution.

[0046] According to the present invention, the dilute acid is dilute hydrochloric acid, and the concentration is preferably 9.5%-10.5%.

[0047] According to the present invention, the neutralization step includes neutralizing to pH 4-4.5 with dilute acid.

[0048] The present invention further relates to a method for treating cancer by combining the above-described photosensitizer composition with photodynamic therapy, or the use of the above-described photosensitizer composition in the preparation of a medicament for photodynamic therapy of cancer or other diseases.

[0049] Beneficial effects

[0050] Photosensitizers prepared by using a certain amount of low molecular weight chitosan sodium salt and other ingredients in a prescribed ratio can not only guarantee consumer demand for a long period of time, but also prepare intravenous injections for medical use, simplifying medical applications in medical institutions. Furthermore, deacetylated chitosan is also a cholesterol-lowering drug.

[0051] The method of this invention treats methyl methacrylate (a-methyl methacrylate) soluble in acetone with an alkaline aqueous solution in an ultrasonic bath at 38-42℃ (frequency 20-80 kHz). No other impurities are generated during the hydrolysis process, and the components of the reaction mixture are not oxidized (low-temperature treatment, fewer impurities, and high purity).

[0052] This invention improves the stability of photosensitizer lyophilized agents by optimizing the ratio between dihydroporphyrin E6 or pharmaceutically acceptable salts and stabilizers; the stabilizing components in the photosensitizer are kept to a minimum; and a certain amount of deacetylated chitosan (chitosan), which helps lower cholesterol, is added to the composition of the photosensitizer. Furthermore, this invention also optimizes the method for preparing a dihydroporphyrin E6 solution with a purity of over 98% from methyl pheophylate (different stabilizers, smaller amounts, improved stability). Detailed Implementation

[0053] The technical solution of the present invention will now be clearly and completely described in conjunction with specific embodiments thereof. Obviously, the described embodiments are only a part of, and not all of, the present invention. All variations obtained by those skilled in the art based on the embodiments of the present invention without inventive effort fall within the scope of protection claimed by the present invention.

[0054] Example 1 (Preparation Example)

[0055] In an environment where argon gas vigorously generates bubbles, 500 ml of 10% sodium hydroxide solution is added to 800 ml of acetone solution containing 4 g of methyl pheophylate a within 1 hour. The mixture is then stirred for 2 hours in an ultrasonic bath at 40°C (25 kHz ultrasonic frequency). After cooling to 5-10°C, 2 L of distilled water is added. The solution is then neutralized to pH 4-4.5 with dilute hydrochloric acid (1:3) under an environment where argon gas vigorously generates bubbles, causing the precipitate to disperse. The upper layer of the solution will show a distinct light purple color. The upper layer should be clarified, while minimizing the amount of precipitate suspended in the solution. The solution is then filtered through an 8 cm diameter glass filter using 5 cm high diatomaceous earth 545. The diatomaceous earth is washed with deionized water until no inorganic salts are present in the filtrate. The surface of the diatomaceous earth 545 contains dihydroporphyrin E6, which is separated and placed in a flask. Add 150 ml of 1% meglumine solution and 5% chitosan (molecular weight 1,000 Da) solution to pyrogen-free water, stir and mix for 5 minutes, and filter the solution through a diatomaceous earth 545 filter to obtain dihydroporphyrin E6 solution. After filtration, add another 250 ml of 1% meglumine solution and 5% chitosan solution to the pyrogen-free water. Further rinse the diatomaceous earth with 600 ml of pyrogen-free water, and filter the solution twice through a top-mounted filter (0.22 nm micropores), adjusting the total volume of the solution to 1 L. If necessary, adjust the pH of the solution to 9.25-9.3. Pour into 10 ml dark glass vials and freeze-dry under standard conditions to obtain 100 vials of loose, porous photosensitizer freeze-dried powder for injection. Each vial contains 37.5-40 mg of the main substance, namely dihydroporphyrin E6 (or not less than 75-80 mg of dihydroporphyrin meglumine salt). At a wavelength of 655 nm and a solution pH of 9.0-9.5, the optical density of the photosensitizer in the dihydroporphyrin gel in the glass bottle is 225-235 / ml. Similar photosensitizers, such as FOTODITAZINA, have an optical density OD (at 655 nm) of 205-210 / ml, and RADA KhLORINA has 175-185 / ml.

[0056] Example 2 (Preparation Example)

[0057] In an environment where argon gas vigorously generates bubbles, 500 ml of 10% sodium hydroxide solution is added to 800 ml of acetone solution containing 4 g of methyl pheophylate a within 1 hour. The mixture is then stirred for 2 hours in an ultrasonic bath at 40°C (25 kHz ultrasonic frequency). After cooling to 5-10°C, 2 L of distilled water is added. The solution is then neutralized to pH 4-4.5 with dilute hydrochloric acid (1:3) under an environment where argon gas vigorously generates bubbles, causing the precipitate to disperse. The upper layer of the solution will show a distinct light purple color. The upper layer should be clarified, while minimizing the amount of precipitate suspended in the solution. The solution is then filtered through an 8 cm diameter glass filter using 5 cm high diatomaceous earth 545. The diatomaceous earth is washed with deionized water until no inorganic salts are present in the filtrate. The surface of the diatomaceous earth 545 contains dihydroporphyrin E6, which is separated and placed in a flask. Add 150 ml of 1% meglumine solution and 5% chitosan (molecular weight 30,000 Da) solution to pyrogen-free water, stir and mix for 5 minutes, and filter the solution through a diatomaceous earth 545 filter to obtain dihydroporphyrin E6 solution. After filtration, add another 250 ml of 1% meglumine solution and 5% chitosan solution to the pyrogen-free water. Further rinse the diatomaceous earth with 600 ml of pyrogen-free water, and filter the solution twice through a top-mounted filter (0.22 nm micropores), adjusting the total volume of the solution to 1 L. If necessary, adjust the pH of the solution to 9.25-9.3. Pour into 10 ml dark glass vials and freeze-dry under standard conditions to obtain 100 vials of loose, porous photosensitizer freeze-dried powder for injection. Each vial contains 37.5-40 mg of the main substance, namely dihydroporphyrin E6 (or not less than 75-80 mg of dihydroporphyrin meglumine salt). At a wavelength of 655 nm and a solution pH of 9.0-9.5, the optical density of the photosensitizer in the dihydroporphyrin gel in the glass bottle is 225-235 / ml.

[0058] Example 3 (Effect Example)

[0059] Application conditions for high-performance liquid chromatography (HPLC): Prominence LC-20 chromatograph (Japan), LC-20AD pump, SRT-20A thermostat, SPD-20A detector-spectrophotometer, PHENOMENEX "LUNA" series column C-18, 4×250 mm, 5 μm, eluent Меон:НЂо:CF3COOH=90:10:0.01; flow rate 0.85 ml / min; volume 5 μL; temperature 25℃; detection at 405 nm wavelength.

[0060] A 10 μL solution of dihydroporphyrin was dissolved in 1 mL of eluent to form an acidic solution with a color change from brown to blue, indicating the formation of the divalent cation e6 of dihydroporphyrin. The resulting solution was filtered through a disposable 0.45 μm syringe filter (CHROMAFIL) before analysis.

[0061] A sample of dihydroporphyrin E6 lyophilized powder can be used as a standard. During the storage of the photosensitizers in Examples 1 and 2 of this invention, the resulting reaction mixtures showed virtually no change. The concentration of the solution decreased by only 0.3%-0.5% over one year, which is higher than the stability of all currently known pharmaceutical photosensitizers on the market.

[0062] The following table shows the comparison results of the long-term stability test content determination (wt%) after adding different stabilizers:

[0063]

Claims

1. A photosensitizer composition comprising dihydroporphyrin e6-triglucamine salt and a stabilizer, wherein the stabilizer comprises meglumine and low molecular weight chitosan, wherein the content of each component by weight percentage is 1-1.1% dihydroporphyrin e6-triglucamine salt, 0.4-0.5% meglumine, and 0.09-0.11% low molecular weight chitosan, wherein the low molecular weight is 600-50,000 Da, and wherein the photosensitizer composition is prepared by a method comprising the following steps: (1) In 38-42 o In the ultrasonic bath of C, the organic solvent solution of methyl pheophylate a was treated with an alkaline aqueous solution. (2) Neutralize with dilute acid. (3) The precipitate of dihydroporphyrin E6 was separated by diatomaceous earth filtration. (4) Dihydroporphyrin E6 was extracted from diatomaceous earth by adding meglumine and a low molecular weight chitosan aqueous solution, and (5)Optionally freeze-dry the extracted dihydroporphyrin E6.

2. The photosensitizer composition of claim 1 is a lyophilized formulation.

3. The photosensitizer composition of claim 1, wherein the alkaline aqueous solution is selected from NaOH or KOH aqueous solution.

4. The photosensitizer composition of claim 1, wherein the frequency of the ultrasound is set to 20-80 kHz.

5. The photosensitizer composition of claim 1, wherein the organic solvent solution of methyl pheophylate a is an acetone solution.

6. The photosensitizer composition of claim 1, wherein the dilute acid is dilute hydrochloric acid.

7. The photosensitizer composition of claim 1, wherein the neutralization step comprises neutralizing with dilute acid to pH 4-4.

5.

8. Use of the photosensitizer composition according to any one of claims 1-7 in the preparation of a medicament for photodynamic therapy of cancer or other diseases.

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