EA002318B1 - Method for preparing pharmaceutical medicament for photodynamic therapy of cancer diseases - Google Patents

Method for preparing pharmaceutical medicament for photodynamic therapy of cancer diseases Download PDF

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Publication number
EA002318B1
EA002318B1 EA199800948A EA199800948A EA002318B1 EA 002318 B1 EA002318 B1 EA 002318B1 EA 199800948 A EA199800948 A EA 199800948A EA 199800948 A EA199800948 A EA 199800948A EA 002318 B1 EA002318 B1 EA 002318B1
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EA
Eurasian Patent Office
Prior art keywords
solution
chlorin
chlorophyll
extraction
drug
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EA199800948A
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Russian (ru)
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EA199800948A1 (en
Inventor
Людмила Васильевна Пленина
Станислав Валентинович Хлюстов
Людмила Александровна Грубина
Георгий Афанасьевич Кочубеев
Эдвард Антонович Жаврид
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Научно-Производственное Республиканское Унитарное Предприятие "Диалек"
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Priority to EA199800948A priority Critical patent/EA002318B1/en
Publication of EA199800948A1 publication Critical patent/EA199800948A1/en
Publication of EA002318B1 publication Critical patent/EA002318B1/en

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Abstract

A method of obtaining a pharmaceutical preparation for photodynamic therapy of oncological diseases includes the CO-extraction and separation of vegetable carotenoids and fatty acids from plant materials under a pressure of 10-300 bar, extraction of chlorophyll and its derivatives with an organic solvent, their oxidation with the release of feofitin and its subsequent transformation, and the conversion of chlorophyll carry out immediately by processing the extract with concentrated hydrochloric acid, and after its oxidative cleavage and separation from hydrochloric acid p Solution and precipitation with alkali at pH 4.0 ± 0.2 carry out the purification of the preparation and inject a stabilizer, a solution of chitosan.

Description

The invention relates to the pharmaceutical industry and relates to a method for producing a pharmaceutical preparation used in medicine for photodynamic therapy of cancer.
Known methods for producing photosensitizing preparations from plant materials by extracting fresh or specially dried raw materials with polar solvents (alcohol, acetone) and obtaining an extract of the sum of chlorophylls a and b. The sum of the pheophytins a and b is precipitated from the chlorophyll extract, from which pure pheophytin a is isolated by chromatographic purification. Unstable chlorin is obtained from pheophytin a by oxidation in alcohols (methyl, ethyl, propyl) containing alkali, and its processing by heating and boiling in organic solvents or decarboxylating agents [1].
However, these methods are complex, multi-stage and have a low yield of the target product.
A known method of obtaining a pharmaceutical preparation for photodynamic therapy, modified at the stage of purification of the target product [2].
The method consists in the fact that chlorophyll derivatives are isolated from plant material by stagewise treatment with an organic solvent. First, a mixture of chlorophylls a and b is isolated by extraction of raw materials with a polar solvent (ethanol), from which pheophytins a and b are precipitated using the Girard reagent. Next, pure pheophytin is obtained from this mixture, and by chromatographic purification on silica gel, pheophytin is then subjected to oxidative cleavage in diethyl ether with the addition of 20-30% potassium hydroxide to it by bubbling with air at room temperature for 2 h until unstable chlorine is formed, which then process as described above.
However, this method is also multi-stage, time-consuming and not suitable for industrial production. The yield of the target product is not high due to the loss of chlorophyll derivatives at the stage of processing the mixture with Girard reagent and chromatographic purification on silica gel, as well as during extraction with diethyl ether from a slightly acidic medium, in which part of the chlorin does not transform into an acidic compound, but remains in the aqueous phase. The known method of producing chlorin in an industrial environment is not technologically advanced, since it requires the use of a large amount of explosive and harmful solvent - diethyl ether, contains a significant number of labor-intensive extraction processes and requires large volumes of raw materials. In addition, during the extraction with ethanol, chlorophyll undergoes allomerization, i.e. Under these conditions, partial oxidation of the isocyclic pigment ring may be observed. When saponifying pheophytin with an oxidized isocycle, the main reaction product is not chlorine, but a series of chlorin-like products, such as purpurins.
The problem to which the invention is directed, is to simplify the method of obtaining a pharmaceutical preparation, prevent oxidation of the chlorophyll isocycle, increase the yield of the target product.
The problem is solved in that in the method for producing a pharmaceutical preparation by extracting chlorophyll and its derivatives from an organic plant with an organic solvent and oxidative cleavage with the release of pheophytin and its subsequent conversion into chlorine, dried nettle, lupine, alfalfa and spirulina leaves are used as a raw material, and the extraction is carried out in two stages. First, a mixture of carotenoids and fatty acids is isolated by CO 2 extraction from raw materials at a pressure of 10-300 bar, and then chlorophyll derivatives are extracted with ethanol, immediately treated with concentrated hydrochloric acid to form a precipitate of pheophytin and dissolved in an alcohol solution in vacuum.
The oxidative cleavage of phytol and the opening of the isocycle in an alcohol solution of alkali are carried out. Then it is dissolved in acid and a precipitate is formed of sodium hydroxide, consisting mainly of chlorin, which is subjected to stabilization, drying and purification from impurities by dissolving in a minimum amount of acetone.
What is new according to the invention is that plant materials are subjected to CO 2 extraction under pressure to extract carotenoids and fatty acids. And after the extraction of carotenoids and fatty acids from the plant mass, chlorophyll derivatives are isolated by extraction with a polar solvent ethanol. The alcohol extract of the pigments is immediately treated with concentrated hydrochloric acid with the release of chlorin. Such processing of plant materials allows you to extract chlorophyll derivatives without loss and, thereby, increase the yield of pheophytins.
And the treatment of pheophytins with concentrated hydrochloric acid makes it possible to more fully separate chlorin from a precipitate of chlorophyll pigments in a solution.
The method is as follows.
Plant materials are dried in a stream of air at 40-80 ° C.
Dehydrated raw materials are placed in an extractor for CO2 extraction and extraction of carotenoids and fatty acids is carried out under a pressure of 10-300 bar and a temperature of 25 ° C. The grass mass is discharged from the CO2 extractor, loaded into a vessel, poured with ethanol, and chlorophyll is extracted. After extraction, the ethanol solution of chlorophyll a + b is filtered through a glass filter. Concentrated hydrochloric acid was immediately added to the extract at the rate of 2 ml per 1 liter of extract, and after 12 hours the amount of pheophytins a + b was precipitated as a black precipitate, which was filtered off and washed repeatedly with alcohol. Pheophytin a + b is dissolved in a minimum amount of acetone, and then alcohol in a vacuum. Then, an alcoholic KOH solution is added to the reaction vessel under vacuum to a concentration of 15%. As a result, saponification of phytol and opening of the isocycle occurs. The reaction mixture is then poured into atmospheric pressure in four times the amount of water to terminate the saponification reaction.
The alkaline solution is acidified with hydrochloric acid to a content of 4.7% in the total solution. In this case, the dissolution of chlorin in acid occurs. Then, the solution is neutralized with a 30% sodium hydroxide solution, and a precipitate is obtained consisting of a number of pigments, excluding chlorin. The precipitate is separated, and the solution is filtered, and a precipitate with a content of chlorin of 5060% falls out of the solution during neutralization, which is washed, collected and dried at a temperature of + 37 ° C.
To clean chlorin from impurities, it is dissolved in a minimum amount of acetone. The insoluble part of the preparation is separated by centrifugation, the solution is diluted with distilled water 1: 1 and the pH is adjusted to 3.4, while the precipitate formed is separated by centrifugation under the same conditions. The precipitate is collected and dried in a thermostat at a temperature of + 37 ° C.
It was found spectrophotometrically that after the cleaning procedure, the content of the active substance in the preparation increases by 10-15%.
Purified chlorin is dissolved in water, sterile filtered. To stabilize the drug, a solution of chitosan is introduced, which reduces general toxicity and increases the specific properties of the drug. Then the solution is poured into vials and freeze-dried from the frozen state. The dried preparation is capped under sterile conditions and rolled up.
The described method for producing a pharmaceutical preparation for photodynamic therapy is convenient in that, in addition to the use of readily available reagents, it does not contain technologically complex operations. In addition, the main solvent, ethyl alcohol, after separation of pheophytins can be distilled and used repeatedly in this technology.
Introduction stabilizer chitosan allows you to get the drug, not only in dry but also in liquid form.
In accordance with the invention, the resulting preparation contains freeze-dried chlorin in the form of trisodium salt.
Physico-chemical characteristics of the drug are as follows.
Molecular Weight of 662 Yes. The preparation contains at least 0.3 mg of chlorin per 1 mg of dry matter. The rest is sodium chloride, necessary for freeze drying of the drug, and a stabilizer.
In appearance, the drug is a black lyophilized amorphous powder with a greenish-violet metallic sheen. Without smell. The pH of the aqueous solution is determined potentiometrically. The contents of 1 vial, dissolved in 20 ml of water, should have a pH of 8.5 ± 0.5.
An aqueous solution of 0.002 g of the drug in 400 ml of water should be transparent.
The drug is sparingly soluble in pyridine (1 g in 100 ml), in water (1 g in 100 ml), in a 0.9% isotonic sodium chloride solution (1 g in 100 ml).
Authenticity is determined spectrophotometrically from the absorption spectrum at wavelengths from 400 to 700 nm.
Chlorin has a characteristic spectrum in the form of four main peaks at 401.5, 500, 530, 560, 605, 665 nm (Fig. 1).
As a second test for authenticity, thin-layer chromatography on silofol plates — IU-256 — is used.
As a solvent, hexane, acetone, chloroform in a ratio of 1: 1: 1 are used. On a chromatographic plate, 2 μl of a solution of chlorin in water is applied. At the end of the chromatography process and drying of the plate, one bright green spot at the start and up to seven slightly colored spots with K. are detected on the chromatogram. I = 0.08 ± 0.03; K. E = 0.2 ± 0.05; B (s = 0.31 ± 0.07; B u = 0.44 ± 0.09; B G5 = 0.60 + 0.10; B G6 = 0.85 ± 0.07; Β ί7 = 0, 95 ± 0.04.
The test for purity and permissible limits of impurities is carried out according to GF XI, no. 1, p. 165.
The content of heavy metals is determined by the method of wet mineralization according to the article Kompedium Medikumatorum.
The copper content in the preparation does not exceed 1.2 × 10 -3 %, zinc 8.3 × 10 -3 %, iron 1 × 10 -1 %, magnesium 1.25 × 10 -2 %, aluminum 1.3 × 10 -2 %, molybdenum 0.17%.
The preparation does not contain cobalt, mercury, lead, strontium, arsenic.
Organic impurities in the preparation are absent.
The residual moisture of the drug should not exceed 12%.
Quantification of chlorin is based on spectrophotometric measurement of the optical density of the solution at 665 nm.
The calculation of the content of chlorin in the preparation is carried out according to the formula
C = Bx50 / Ex1x 662, where Ό is the optical density of the solution at 665 nm;
E is the molar extinction coefficient of chlorin in a given solvent at a given wavelength of 5.3x10 -4 M -1 cm;
- the thickness of the layer of the cell (in cm);
662 — molecular weight of the trisodium salt of chlorin;
- coefficient of dilution of the drug.
The content of chlorin in the dry preparation should be 50 ± 5 mg.
Example 1 (substrate - nettle).
I. Obtaining pheophytins.
The nettle leaves, crushed to powder, (3.5-4 kg) are soaked in 20 l of a mixture of water with alcohol in a ratio of 1: 1 for 1.5-2 hours at a temperature of 15-25 ° C. Then the water is suctioned off under vacuum. The dehydrated crop is placed in a CO 2 extractor and the carotenoids and fatty acids are extracted under a pressure of 10-300 bar. The herbal mass is removed from the CO 2 extractor, placed in a glass tube (L = 120 cm, Ό = 40 mm) and 15 l of ethanol is poured for 1.5-2 hours. The extraction is repeated 3 times. The extracts are combined, filtered through a glass, paper or cotton-gauze filter.
Concentrated HCl (2 ml per 1 L of extract) was immediately added to the extract, after 12 hours the precipitate was filtered off and dried in air.
Yield - 30 g of powder.
II. Chlorin synthesis.
g of pheophytin powder is dissolved in 250 ml of acetone. The resulting solution is mixed with 5 l of alcohol. The mixture is aerated with nitrogen for 3040 min at a temperature of + 40 ° C.
In parallel, an alcoholic solution of potassium hydroxide is prepared at the rate of 1.2 kg per 5 l of 96% alcohol in a nitrogen atmosphere. Then the alkali solution is pumped into a container with pheophytin dissolved in ethanol.
The reaction is carried out for 40-50 minutes at a temperature of + 40 ° C in a nitrogen atmosphere.
At the end of the reaction, the solution is neutralized with 28 l of a previously prepared solution of 9% hydrochloric acid to pH = 0.4-0.8 with vigorous stirring. This precipitates, consisting of impurity pigments, which are separated by filtration through the pulp on the nut filter.
The pH of the filtrate was adjusted to 4.0 ± 0.2 with a 30% sodium hydroxide solution. The precipitate, consisting mainly of chlorin, is collected in a centrifuge and washed with distilled water to pH = 3.6.
The resulting precipitate is dried at a temperature of + 37 ° C to constant weight.
The product yield is about 5 g.
III. Purification from impurities.
Take a sample of the drug and dissolve in a minimum amount of acetone. The insoluble part of the preparation is separated by centrifugation at 3000 rpm for 15 minutes.
Then the solution was diluted with distilled water 1: 1 and the pH was adjusted to 3.4, while a precipitate formed which was separated by centrifugation under the same conditions. The precipitate is collected and dried in a thermostat at a temperature of + 37 ° C.
IV. Drug stabilization.
The resulting dry substance is dissolved in distilled water at pH = 11.0. The resulting solution was filtered through a paper filter. The pH of the filtrate is adjusted to a value of 8.08.5 and 125 ml of stabilizer are introduced (dissolved 2% chitosan solution). Next, the resulting solution is sterile filtered, poured into vials and dried leophilically.
Humidity of the resulting drug is not more than 10%.
Example 2 (substrate - lupine).
I. Obtaining pheophytins.
The scheme for obtaining pheophytins from lupine is similar to the scheme for obtaining from nettle as in example 1.
The product yield is about 50 g.
II. Chlorin synthesis.
g of pheophytin is dissolved in 0.5 l of acetone. Next, see the scheme for the production of chlorin and purification from impurities as in example 1.
Example 3 (the substrate is a mixture of nettle, lupine, alfalfa in a weight ratio of 1: 1: 1) obtaining a liquid form.
I. Obtaining pheophytin.
A plant mixture of 80 kg, dried at a temperature of 40-80 ° C in a stream of air, is soaked in 300 l of a mixture of water with ethanol (1: 1) for 1.5 hours at a temperature of 15-25 ° C. Then the solution is suctioned off under vacuum. The dehydrated grass is placed in an extractor for CO2 extraction under a pressure of 10-300 bar and a temperature of 25 ° C. Then the grass mass is discharged from the CO2-extractor, loaded into a vessel for processing with ethanol and 3 extraction of chlorophyll with ethanol (200-250 l each) is carried out. The extracts are combined and then the preparation of pheophytin is carried out according to the scheme for obtaining nettle extract from Example 1.
The product yield is 800 g.
II. Getting chlorin.
The resulting pheophytin is dissolved in 2 l of acetone. The resulting solution was poured into 15 l of ethanol for 45-60 minutes, a solution of KOH in ethanol (3.5 kg per 15 l of alcohol) was added. The reaction lasts 60 minutes at a temperature of + 40 ° C in a nitrogen atmosphere.
The solution was neutralized with 9% HC1 (15 L per 60 L H2O), pH = 0.6-75 L.
The precipitate formed is removed on a nut filter. The pH of the filtrate was adjusted to 4.0. The precipitate formed is collected in a centrifuge and washed with acidified water.
The product yield is about 100 g.
The substance is dissolved in distilled water at pH = 11.0.
The resulting solution was filtered through a paper filter. The pH of the filtrate is adjusted to a value of 8.0-8.5 and a stabilizer, 2% chitosan solution, is introduced.
The resulting solution is sterile filtered and poured into vials.
The pharmaceutical preparation obtained by the proposed method is available in dry form at 50 ± 100 mg in 50 ml vials or in liquid form. The content of the active substance of chlorin is at least 0.3 mg / mg dry matter.
By its action, the drug refers to photosensitizers that selectively accumulate in tumor tissue. Subsequent exposure to light with a wavelength of 665 nm leads (photodynamic therapy) to the excitation of a chlorin molecule and the subsequent destruction of tumor tissue. The effect of photosensitization is manifested in impaired cellular respiration, protein synthesis, inhibition of glycolysis, and in a change in the permeability of the cell membrane. Its use in the treatment of tumors is based on these properties of chlorin. The introduction of a stabilizer reduces general toxicity and increases its specific properties.
The drug is administered intravenously. A solution of the drug is prepared ex 1trog. The contents of the vial are diluted in 50 ml of physiological solution of sodium chloride. A therapeutic dose of the drug is injected into a vein for 20 minutes in 200 ml of physiological saline.
The resulting photosensitizing agent passed clinical trials and showed positive results.
Clinical trials of the pharmacological properties of the chlorin photosensitizer were carried out during the photodynamic treatment of tumors in mice and rats by intravenous administration of a photosensitizer and subsequent irradiation of tumor foci.
The chlorin photosensitizer has a maximum absorption at a wavelength of 660 nm. Therefore, a laser with the indicated wavelength of light was used for photo irradiation of tumors.
Tests have shown that the drug is a low-toxic substance. So, for bE mice | 0 is 40 mg / kg, for rats - 30 mg / kg.
Inverted tumors of rats and mice are characterized by a slower accumulation and excretion of the drug compared to normal tissues.
24-72 hours after administration, the concentration of chlorin in tumors exceeded its content in the liver, kidneys, spleen and muscles in animals with sarcoma 45, respectively, 2.6, 1.2, 4.3 and 6.5 times; with sarcoma M-1 - 4.6, 2.1, 7.7 and 11.5 times; with sarcoma 180 - 6, 12, 10 and 55 times; with a solid Ehrlich tumor - 6.5, 13, 11 and 65 times.
The range of therapeutic doses of chlorin was 1-10 mg / kg. The depth of damage to the neoplasms when using the drug in these doses varied from 4 to 16 mm, and the antitumor effect of the photodynamic effect was directly dependent on the concentration of the photosensitizer in the blood, tumor tissue, and on the energy exposure of light exposure.
Different types of tumors differed in sensitivity to photodynamic effects: the cure rate of animals with alveolar liver cancer RS-1, sarcoma 45, sarcoma M-1, and Pliss lymphosarcoma was 100, 100, 50, and 20%, respectively.
The photodynamic effect with chlorine causes structural and functional changes and necrosis of the tumor tissue. Depending on the dose of the drug, the death of tumor cells can occur as a result of a direct photocytotoxic effect or indirectly due to irreversible circulatory disorders followed by autolysis.
The main side effect of chlorin was expressed in minor structurally functional disorders in the liver with prolonged (within 30 days) administration of the drug. With 2-3 times the introduction of pathological changes were not observed.
In toxic doses causing the death of all or part of the experimental animals, pronounced morphological changes in the liver were observed.
The maximum damage to the tumor and normal tissues was observed during photo-irradiation at the moment of the highest concentration of chlorin in the blood, and the predominant damage to the tumor tissue was observed during the time interval between administration of the drug and the exposure to light for 24-72 hours.The therapeutic dose of photo-irradiation is 45-135 J / cm 2 .
The test results showed that the pharmaceutical preparation obtained by this method is an effective photosensitizer for the treatment of cancer using the laser-dynamic therapy method. This compound is superior in its characteristics to the currently used preparations based on hematoporphyrin.

Claims (2)

  1. CLAIM
    1. A method of producing a pharmaceutical preparation for the photodynamic therapy of cancer, including extraction of chlorophyll and its derivatives with an organic solvent from plant materials, their oxidation with the release of pheophytin and its subsequent conversion, characterized in that, from plant materials by CO2 extraction under a pressure of 10-300 bar, carotenoids and fatty acids are separated before chlorophyll is extracted with ethanol, and the conversion of chlorophyll is carried out immediately by treatment with concentrated hydrochloric acid minutes and after oxidative cleavage and release of the hydrochloric acid solution and deposition of the alkali whose pH = 4.0 ± 0.2 is carried out subsequent purification and formulation administered stabilizer.
  2. 2. The method according to claim 1, characterized in that a solution of chitosan is used as a stabilizer.
EA199800948A 1998-09-03 1998-09-03 Method for preparing pharmaceutical medicament for photodynamic therapy of cancer diseases EA002318B1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03215433A (en) * 1990-01-19 1991-09-20 Ichimaru Pharcos Co Ltd Anti-mutagenic action substance originated from hydrangeae dulcis folium
DE4111208A1 (en) * 1991-04-06 1992-10-08 Inge Witte Plant extract for treating cancer - comprises Thuja soln., elder, beetroot, wheat germ oil, lacto- and soya protein and wild yeast
JPH05186361A (en) * 1991-03-04 1993-07-27 Kobe Steel Ltd Carcinostatic substance and its production
RU2078578C1 (en) * 1995-02-23 1997-05-10 Центр народной медицины "Юнона" Agent and method of antitumor activity increase and cytostatic therapy by-side effects decrease

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03215433A (en) * 1990-01-19 1991-09-20 Ichimaru Pharcos Co Ltd Anti-mutagenic action substance originated from hydrangeae dulcis folium
JPH05186361A (en) * 1991-03-04 1993-07-27 Kobe Steel Ltd Carcinostatic substance and its production
DE4111208A1 (en) * 1991-04-06 1992-10-08 Inge Witte Plant extract for treating cancer - comprises Thuja soln., elder, beetroot, wheat germ oil, lacto- and soya protein and wild yeast
RU2078578C1 (en) * 1995-02-23 1997-05-10 Центр народной медицины "Юнона" Agent and method of antitumor activity increase and cytostatic therapy by-side effects decrease

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