DE3744571A1 - Pharmaceutical compositions for stimulating the immune system and process for their production - Google Patents

Abstract

Pharmaceutical composition for stimulating the immune system, containing as active ingredients isobutylamides of polyunsaturated carboxylic acids with a chain length of from 10 to 14 carbon atoms, where the isobutyl radical can optionally be substituted by lower alkyl groups, as well as alkenes and alkynes which have chain lengths of, in each case, from 10 to 18 carbon atoms and carry a carbonyl group in position 2.

Description

The invention relates to pharmaceutical preparations with immunostimulating Effect and process for their manufacture.

Immune stimulation as a therapeutic concept has been in medicine since known for a long time. In general, this means the administration of Substances that have little or no antigenic activity have, but are able to point to non-specific or specific way to induce the body's defense mechanisms. According to current knowledge, a variety of substances is able to To stimulate immune defense, in particular various minerals to Example Al (OH) ₃, MgSO₄, beryllium, vegetable oils with or without additives of mycobacteria as well as a number of herbal active ingredient groups such as Alkaloids, sesqui and diterpene compounds, quinones and polysaccharides too are called. The entire complex of immune stimulation is described in detail from Z. B. Chedid, L., et al. Immune stimulation, Springer Verlag, Heidelberg / New York 1980; Hedelberger, M., Structure and Immunological Specifity of polysaccharides; Progress d. Chem. Org. natural 42, 288 (1982); Drews, H., Possibilities of Immune Stimulation, Swiss Pharma 2, 9, (49) (1980).

The exact mode of action of immunostimulating substances could in most Cases have not yet been finally clarified. Generally these show Substances have an influence on the proliferation of the immune competent Cells, but they do not leave a memory reaction. This means that as targets for the immunostimulating substances the macrophages and granulocytes as well as T and B lymphocytes in the foreground stand. The effect can be direct or indirect, e.g. B. about that Complement system or via lymphocytes, via the production of interferon or lysomal enzymes (e.g. lymphokines, colony stimulating factor and others), as well as an increase in macro- and microphagocytosis. Because of the integration of non-specific and specific defense mechanisms is always with cascade effects and simultaneous influencing multiple defense systems.  

In medicine, there are preferred areas of application for immune stimulation especially the therapy of mixed infections, chronic, persistent, chemotherapy-resistant, bacterial and viral infections, the prophylaxis of opportunistic infections in high-risk patients, the therapy of malignant diseases and to a certain extent also the treatment of autoimmune diseases. Immune stimulants can also be used in the Cytostatics therapy for partial compensation of the associated Immunosuppression can be used.

In this context, Echinacea preparations are becoming increasingly larger Gained meaning. In the pharmaceutical substance list (7th edition, advertising and sales company Deutscher Apotheker mbH, Frankfurt / Main 1986) and the list of preparations in naturopathy (6th year, Sommer Verlag GmbH, Teningen 1986) approx. 230 preparations are listed, the preparations from Echinacea purpurea, Echinacea angustifolia or Echinacea pallida. Echinacea angustifolia and Echinacea pallida are mainly ethanolic / watery Extracts from the roots used. In contrast, at Echinacea purpurea used both the herb and the roots.

The main areas of application for Echinacea-containing preparations are flu Infections, inflammation, chronic polyarthritis, bladder disorders, poorly healing wounds and adjuvant tumor therapy. successes in the prophylactic and therapeutic use of stimulation attributed to the body's defenses, cf. G. Harnischfeger and H. Stolze in Notabene Medici, 10th year, November 1980, 11:484.

The polysaccharides are an immunologically active ingredient group of Echinacea known, cf. Wagner et al., Drug Research 35, 1069 (1985). These authors isolated and characterized two polysaccharides from E. purpureae herba, which is a 4-0-methyl glucurono-arabinoxylan with a molecular weight of approximately 35,000 D, and a Arabinorhamnogalactan with an average molecular weight of approx. 450,000 D. acts. They stimulate phagocytosis of granulocytes in vitro and in vivo and activate macrophages towards increased cytotoxicity Tumor cells. In the commercially available aqueous-ethanolic extracts  these polysaccharides are not included due to their ability to precipitate with alcohol. With their presence is therefore only in squeezed juices or in extracts to be expected, which are produced with a very high water content.

The disadvantages of the Echinacea preparations that have been commercially available so far are especially in that they contain only minor and fluctuating ingredients Shares included, which is why they cannot be chemically standardized exactly Prepares a targeted therapeutic use with reproducible results not allow.

Surprisingly, herbal active ingredient groups have now been found that an increase in immune defense, in particular an increase in the phagocytosis rate of the monocytic system. These drug classes are chemically characterized as carboxylic acid isobutylamides of polyunsaturated Carboxylic acids with a chain length of 10 to 14 carbon atoms, wherein the isobutyl radical is optionally substituted by lower alkyl groups can be, as well as alkenes and alkynes with chain lengths of 10 to 18 carbon atoms which carry a carbonyl group in the 2-position.  

The aforementioned isobutylamides include, for example

Undeca- (2E, 4Z) -diene-8,10-diynoic acid- (1) -isobutylamide (1)
Undeca- (2Z, 4E) -diene-8,10-diinoic acid- (1) -isobutylamide (2)
Dodeca- (2E, 4Z) -diene-8,10-diynoic acid- (1) -isobutylamide (3)
Dodeca- (2E, 4E, 10E) -triene-8-in-acid- (1) -isobutylamide (4)
Dodeca- (2E, 4E, 8Z, 10E) -tetraenic acid- (1) -isobutylamide (5)
Dodeca- (2E, 4E, 8Z, 10Z) -tetraenic acid- (1) -isobutylamide (6)
Dodeca- (2E, 4E, 8Z) -triene-acid- (1) -isobutylamide (7)
Dodeca- (2E, 4Z) -diene-8,10-diinic acid-2-methylbutylamide (8)
Undeca- (2E, 4Z) -diene-8,10-diinic acid-2-methylbutylamide (9)
Trideca- (2E, 7Z) -diene-10,12-diinic acid- (1) -isobutylamide (10)

The above-mentioned alkenes and alkynes include, for example, the following Links:

8-hydroxy-tetradeca-9E-en-11,13-diin-2-one (11)
8-hydroxy-pentadeca-9E-en-11,13-diin-2-one (12)
8-hydroxy-pentadeca-9E, 13Z-dien-11-in-2-one (13)
Tetradeca-8Z-en-11,13-diin-2-one (14)
Pentadeca-8Z, 13Z-dien-11-in-2-one (15)
Pentadeca-8Z, 11Z-dien-2-one (16)
Pentadeca-8,11,13-trien-2-one (17)
Pentadeca-8-en-2-one (18)
Pentadeca-8-en-11,13-diin-2-one (19)
Heptadeca-8,11-dien-2-one (20).

The active ingredient combination according to the invention can be as follows, for example will be obtained:

As a starting material for the production of the above-mentioned isobutylamides roots and / or aerial parts of botanically determined Echinacea purpurea, E. angustifolia and E. pallida plants used while for the recovery of the above alkenes and alkynes in particular the roots of Echinacea pallida are used. Echinacea purpurea and Echinacea pallida can be obtained from the Landesanstalt für Bodenkultur und Plant production Freising / Weihenstephan and Echinacea angustifolia from the Trout Lake Farm, Wa., USA.

Instead of crops from plants, commercial drugs can also be used are used, as far as these were determined botanically. In addition to the Botanical determination can also use the HPLC fingerprints for differentiation be used, with particular reference to falsification must be checked with Parthenium integrifolium.

Manufacturing

Air-dried parts of plants from Echinacea purpurea, Echinacea Pallida and / or Echinacea angustifolia are crushed and then pulverized in a mill. The powder (500 µm sieve) is in a Soxhlet apparatus with an alcohol (up to 4 C atoms) extracted and then concentrated (alcoholic extract). After determination the extract yield, the residue is preferably in 90% Ethanol dissolved. This solution is used to obtain the lipophilic fraction 1: 1 to 1: 3, preferably 1: 1, diluted with water and then at least once, preferably three times, with chloroform or other lipophilic Solvents (e.g. hexane, pentane, dichloromethane, diethyl ether) shaken out. The lipophilic phases are combined, over sodium sulfate dried, evaporated to dryness and the residue is then preferably dissolved in ethanol.  

Alternatively, there is the option of using the powdered plant parts directly with a lipophilic solvent (e.g. chloroform, dichloromethane or hexane) and extract a fraction of the lipophilic To produce ingredients.

In addition, some of the lipophilic ingredients can also be obtained by steam distillation the drug can be obtained ("essential oil"). Therefore z. B. 50 g powdered root drug in a steam distillation apparatus distilled according to DAB 9 with 500 ml of water for 3 hours. The separated essential oil is drained.

The lipophilic parts are produced from the aerial parts of the plant Ingredient fraction, for example, in that the alcohol extract mixed with the same volume of 5% aqueous lead acetate solution and then the precipitated material (chlorophyll, phenols and sterols) is filtered through diatomaceous earth (Celite®). The watery alcoholic filtrate is then mixed with chloroform or another lipophilic solvent shaken out and processed as described above.

The following is the preparation of a lipophilic fraction from Echinacea purpurea roots described using chloroform:

The air-dried whole roots were crushed and then pulverized in a mill. 1,007 g of root powder (500 µm) were in a Soxhlet apparatus for 1 hour with 50 ml of methanol p.a. extracted. After determination the extract yield (0.130 g) was in 10.0 ml of 90% ethanol solved. To obtain the lipophilic fraction, the solution was 1: 1 with water diluted and then shaken three times with 10 ml of chloroform. The chloroform phases were combined, dried over sodium sulfate and the Dry concentrated (13 mg). The residue was dissolved in 2.0 ml of ethanol.

The chloroform fraction was examined for its ingredients using HPLC.

Explanation of the pictures

Fig. 1: Carbon clearance through Echinacea pallida root extracts; Regression coefficient (RC) of coal removal (log E against t) compared to the control.

Fig. 2 shows the separation of the chloroform fraction from different Echinacea species in the HPLC.

E. pallida: 1 = 8-hydroxy-tetradeca-9-en-11,13-diin-2-one; 2 = 8-hydroxy-pentadeca-9-en-11,13-diin-2-one; 3 = 8-hydroxy-pentadeca-9,13-dien-11-in-2-one; 4 = tetradeca-8-en-11,13-diin-2-one; 5 = pentadeca-8,13-dien-2-one; 6 = Penta deca-8,11-dien-2-one.
E. angustifolia: 1, 2, 3, 5 = previously unidentified isobutylamides; 4 = isobutylamide of dodecatetraenoic acid.
E. purpurea: 1 = undeca- (2Z, 4E) -diene-8,10-diinic acid isobutylamide; 2 = Do deca- (2E, 4Z) -diene-8,10-diinic acid isobutylamide; 3 = dodeca- (2E, 4E, 10E) -triene-8-in-acid-isobutylamide; 4 = isobutylamide of dodeca-2,4,8,10-tetraenoic acid.

For the chemical characterization of the Echinacea drug extracts high-performance liquid chromatographic Method used, cf. Bauer et al, German Apoth. Ztg. 127, 1325 (1987) and Bauer et al, Sci. Pharm. 55, 159 (1987).

pharmacology

For the investigation of the influence of lipophilic extracts on the non-specific Immune system test systems were used to test the ability to stimulate of the mononuclear phagocytic system can be measured. For in vivo testing was the Biozzi and Mitarb carbon clearance test, corresponding to that of Wagner et al. published methodology used, cf. Wagner et al. Pharmaceutical Research / Drug Res. 35, 1069 (1985). The Phagocytic activity of mouse liver and spleen macrophages determined. The rate of elimination of injected carbon particles from the Blood compared to a control group treated with isotonic saline. The evaluation is based on the ratio of the slopes of the Regression line of coal elimination (log E against t), where RCtr is the mean Regression coefficient of the substance, and RCc the mean regression coefficient is in control. With a ratio RCtr / RCc <1.5 can strong phagocytosis stimulation is mentioned (DSK grade 2). If this is between 1 and 1.5, it is only a weak stimulation, which is rated with the DSK grade 1. For the detection of oral The extracts were effective in the carbon clearance test using a pharyngeal tube applied. The dosage was of an average dose 50 drops (0.3 to 0.4 mg) of the alcoholic extract three times a day in humans. Since experience has shown that mice are around doses of three times were less sensitive approx. 2 mg extract selected per kg mouse and day. The results are in the table 1 shown.  

Table 1

Results of the carbon clearance test with the different Echinacea root extracts (± hourly; n = 9-15)

For the in vitro investigation of the immunostimulating effect the granulocyte smear test carried out in a modification according to Brandt, cf. Brandt, L., Scand. J. Haematol., Suppl., 2 (1967). The extraction of human Granulocytes were made according to the method described by Wagner et al. described method, cf. Wagner et al., Pharm. Research / Drug Res. 35, 1069 (1985). In this test the phagocytosis ability of human granulocytes in the presence of Yeast particles determined. The results are shown in Table 2, for comparison stimulation with the ethanol extract is shown in Table 3.  

Table 2

Results of the granulocyte smear test with the chloroform fractions of the ethanolic Echinacea root extracts (PI = phagocytosis index; s = standard deviation ( n = 3-6); Stim.PI = stimulation of phagocytosis compared to the control)

Table 3

Results of the granulocyte smear test with the ethanolic Echinacea root extracts (PI = phagocytosis index; s = standard deviation ( n = 3-6); stim.PI = stimulation of phagocytosis compared to the control)

The results of the pharmacological testing of the chloroform fractions, which mainly contain polyacetylenes, isobutylamides and essential oil, compared to the ethanol extract known from the prior art, the following:

The chloroform fraction of the Echinacea pallida extract was found in the carbon Clearance test as far more effective than the entire ethanol extract, cf. Tab. 1 and Fig. 1.

As Tables 2 and 3 show, the chloroform extracts from Echinacea in the concentration range from 10 ^-3 to 10 ^-6 mg / ml test batch are significantly more effective than the ethanol extracts in the in vitro granulocyte smear test. At 10 ^-7 mg / ml there was usually no longer any effect.

The in vitro results correlate well with those in the carbon clearance test achieved in vivo results.

The preparations according to the invention are administered orally or parenterally and can be formulated with the known auxiliaries and carriers.

Claims (10)

1. Pharmaceutical preparation for stimulating the immune system, containing Carboxylic acid isobutylamides with polyunsaturated carboxylic acids a chain length of 10 to 14 carbon atoms, the isobutyl radical can optionally be substituted by lower alkyl groups, as well as alkenes and alkynes with chain lengths of 10 to 18 carbon atoms each, which carry a carbonyl group in the 2-position, if appropriate together with usual auxiliaries and carriers. 2. Pharmaceutical preparation according to claim 1, containing carboxylic acid isobutylamide polyunsaturated carboxylic acids with a chain length from 10 to 14 carbon atoms, the isobutyl radical being substituted by low Alkyl groups can be substituted. 3. Pharmaceutical preparation according to claim 1, containing alkenes and Alkynes with chain lengths of 10 to 18 carbon atoms in the 2-position carry a carbonyl group. 4. A process for obtaining the active ingredients according to one of claims 1 to 3, characterized in that:

• i) crushed dried plant parts of Echinacea purpurea, Echinacea pallida and / or Echinacea angustifolia and then pulverized
• ii) the powder is extracted with an alcohol having 1 to 4 carbon atoms, then the extract is concentrated,
• (iii) dissolving the residue in alcohol, diluting with water, shaking with a lipophilic solvent and
• iv) the lipophilic phases are combined, evaporated to dryness and the residue is taken up in a solvent.

5. The method according to claim 4, characterized in that the roots used as starting material. 6. The method according to claim 4 and 5, characterized in that as Alcohol uses methanol or ethanol. 7. The method according to claim 4 to 6, characterized in that one Residue in step (iii) 1: 1 to 1: 3, preferably 1: 1 with water diluted and at least once, preferably three times, with a lipophilic Shakes out solvent. 8. The method of claim 5 or 7, wherein the lipophilic solvent Chloroform, hexane, pentane, dichloromethane, diethyl ether or mixtures of it is.   9. The method according to claim 4, characterized in that the above-ground Parts of the plant are used, whereby in stage (ii) the alcoholic Extract with the same volume of 5% aqueous lead acetate solution moved and the precipitated material separated before further processing. 10. The method according to any one of claims 4 to 9, wherein the pulverized Plant material extracted directly with a lipophilic solvent becomes.