DE3913040A1 - Use of fungi that produce anthraquinone or di:anthrone dyestuffs - for the manufacture of antiviral preparations - Google Patents

Abstract

The fungi, which are of the Ascomycetes family, produce emodin or scyrin. The prefd variety of fungus is Penicilliopis clavariaeformis solms or a close . An extract (I) of the culture is administered with glycols, glucose syrup glycerine and/or liquid polyethyleneglycol. USE/ADVANTAGE - In the treatment of viral infections such as AIDS, non-A non-B hepatitis viruses, parvovirus etc. The extract can also be used to preserve blood plasma, albumin, clotting factor VIII and IX and immunoglobulin prepns. (I) is easy to produce and can be administered without isolating the active ingredient.

Description

The invention relates to pharmaceutical preparations, in particular especially with an inhibitory effect against viruses and / or retro viruses, and their use.

Viral diseases, especially because of their rapid Spread in the population, a big health Problem. This is especially true for the acquired immune Weak syndrome AIDS (Acquired Immunodeficiency Syndrome). So far it is neither possible to have a vaccine against the the disease causing viruses HIV (Human Immunodeficiency Virus), even after the onset of the disease symptoms to treat the disease sufficiently medicinally. So are although in the meantime some substances in the medical Erpro and partly already in use, the inhibit the multiplication of viruses by converting them of the RNA genome of the viruses in DNA by incorporation of modified Nucleotides abrogate DNA synthesis, thereby degrading the Ver inhibit the growth of viruses. However, these medicines call strong side effects, as well as in healthy human Cells are incorporated the modified nucleotide analogs can and there to cancel a normal and necessary DNA synthesis can lead.

Therefore, there is still a high demand for more Agents for the treatment of AIDS and other viral diseases, e.g. Infections with Non A, Non-B Hepatitis Virus, Parvo Viruses, etc.

Task of the present invention was therefore the Provision of further pharmacologically active preparations that treat both infections and viruses Non A, non-B hepatitis viruses, as well as the treatment of AIDS or ARC (AIDS-related complex, a precursor to AIDS Illness).  

It has now been found that it is using anthra quinone or dianthrone dyes producing fungi is possible to pharmacologically effective in a simple manner Preparing preparations, in particular a pronounced Inhibitory effect on viruses and / or retroviruses show.

An object of the present invention is therefore the Ver use of anthraquinone or dianthrone dyes Fungi for the preparation of pharmaceutical preparations, in particular for the preparation of preparations with a Inhibitory effect on viruses and / or retroviruses.

The fungi used are preferably those which photo Sensitizing and / or fluorescent dyes per duce. In particular, fungi of the family Ascomyceten used, such as Emodin or Scyrin forming Ascomycetes, and primarily Penicilliopsis clavariaeformis solms or its metabolites. But there are also mushrooms like Dermocybe austroveneta, Dermocybe luteostriatula, Dermocybe olivipes, Dermocybe obscureolivea var. brunnea, Dermocybe hypoxanthia, Dermocybe alcalisensibilis, Cortinarius atro virens, Cortinarius ionochlorus, Cortinarius odoratus, Hypomyces aurantius, Hypomyces lactifluorum, Hypomyces trichothecoides, D. alcalisensibilis, Dermocybe alienata and D. luteostriatula in question.

By extraction of these fungi can be pharmacologically effective preparations, in particular with an inhibitory effect on Make viruses and / or retroviruses.

The invention therefore also pharmaceutical Preparations characterized in that they Extracts and / or Ingredients of Anthraquinone or Di  containing or derived from anthrone dyes forming mushrooms consist. In particular, these preparations contain extracts the fungi mentioned above as preferred, and in the first Line of Penicilliopsis clavariaeformis solms.

The pharmaceutical preparations according to the invention can optionally further customary for such preparations Contain carriers and / or additives. An addition of such Substances depends in particular on the type of the desired Form of administration, e.g. as tablets, capsules, pills, syrups, Suspensions, etc., the ent extracts the extracts according to the invention hold.  

In German Patent Application P 38 41 562 of the same Applicant is described a drug which is at least a conjugate of albumin with a compound of the general Contains formula I,

in which R ₁ and R ₂ or / and R ₃ and R ₄ or / and R ₅ and R ₆ or / and R ₇ and R _{8 are} either hydrogen or else R ₁ and R ₂ or / and R ₇ and R _{8 are} each together represent a CC single bond and / or R ₃ and R ₄ together with R ₅ and R _{6 represent} a CC double bond, wherein at least one CC single or CC double bond must be present, R₉ and R₁₀ are each either CH ₃ or -CH (OH) -CH ₃ and R ₁₁ and R _{12 are} each H or a compound of formula II

are ortho to the nitrogen heteroatom the compound of the formula (I) is bonded, in which R 'is H or Acetyl, and optionally other conventional carrier and Contains additives.

It was found that these compounds (conjugate with Albumin) works well in treating various virus diseases, especially AIDS. However, too by other human pathogenic viruses, e.g. Non-A, Non-B Hepatitis or parvo virus-induced diseases be fought.

For coupling a compound of general formula (I) Albumin is this appropriately dissolved in sodium hydroxide solution pH = 13 and with a solution of monomeric human albumin for 30 minutes incubated at 37 ° C. The solution of the compound of formula (I) and the albumin solution (5 g / dl) are in equal parts used. After incubation, buffer with buffered 1 M HCl adjusted to pH 7.0.

Are used e.g. Conjugates with hypericin, pseudo-hypericin, Oxypenicilliopsin, penicilliopsin, protohypericin or / and Fagopyrin.

It has been found that these substances protect against viruses, and especially against HIV viruses have efficacy.

The pharmacological activity was on H 9-III cells an HIV-infected cell line checked by determining the amount of HIV envelope protein p 24 produced on the cell line p24 is hereby using monoclonal antibodies detected by the ELISA technique. The p 24 concentration, which is a measure of AIDS virus production, increases without Addition of the conjugate HC-S (synthetic hypericin) strongly on, but with the addition of the same it falls sharply. Remarkable Here is that the stability of the cells with HC-S addition was not disturbed.  

It has now surprisingly been found that the Invention proper, simple and easy, without the need for an isolie tion of individual ingredients, preparations to be prepared a marked inhibitory effect against viruses and / or retroviruses their inhibitory effect, in particular on HIV, in German Patent Application P 38 41 562.3 described Conjugates are even superior.

The invention therefore also relates to the use of a Pharmaceutical preparation according to the invention for treatment and prophylaxis of viral diseases, as well as for the killing of Viruses and as a virostatic agent in vitro.

Another object is the use of mushroom color substances of anthraquinone or dianthrone dyes Mushrooms for the preparation of pharmaceutical preparations with an inhibitory effect on viruses and / or retroviruses.

It has also been found that a preparation prepared by Ver rubbing of fungal dyes from the anthraquinone and / or dianthrone Type with an extractant for the preparation of standing described extracts of the invention, and in particular especially with glycols, glucose syrup, glycerol and / or liquid Polyethylene glycol is obtained, inhibitory effects on viruses and / or retroviruses with those described above benen pharmaceutical preparations is comparable. against Therefore, the invention is also a pharmaceutical Preparation characterized in that it has a by trituration of fungal dyes from anthraquinone and / or or dianthrone type with glycols, glycose syrup, glycerin and / or liquid polyethylene glycol obtained or consists of it.

The use according to the invention comprises e.g. the usage a preparation according to the invention for prophylactic Ver Delivery to persons at risk of infection, for treatment of blood components, e.g. Preserved blood or blood plasma,  Blood derivatives such as albumin, coagulation factor VIII and IX and Immunoglobulin preparations. For the treatment of preserved blood and blood derivatives are e.g. small quantities of erfindungsge according to the preparation or the inner wall for Storage vessels of such substances with the erfindungsge coated according to the preparation. In the sense of a general Safety should be all in the clinics, blood banks and doctor practice used blood collection tube from the outset so to be secured. This would be a new level of security of the medical and health professionals nals against HIV and other viral infections. except Transfusion medicine could once again be very safe regain the level of trust and confidence of the population.

Anthraquinone or dianthrone dyes forming fungi known from the literature (see, for example, E. Gäumann, Die Pilze, Basel 1949; Steglich and Arnold, Angewandte Chemie 85 (1973) 88f; Steglich et al, Angewandte Chemie 89 (1977) 56f; Giese, Photochem.Photobiol. Photobiol. Rev. 5 (1980) 229-256). To invent According to the invention preferably used fungus Penicilliopsis clavariaeformis Solms cf. e.g. Count of Solms-Laubach, Annales du jardin botanique Buitenzorg VI (1887), page 53; J. Reinke, Annales du jardin bitanique Buitenzorg VI (1887), Page 73; Ch. Dhere, Acad. sci. Paris 197 (1933), page 948, ref. Chem. Zentralblatt 1934, I. Part, page 1894; Ch. Dhere and V. Castelli, C.R. Seanc. Soc. biol. fil. ass. 131, 669, ref. Chem. Zentralblatt 1941, II. Part, page 2331; A.E. Oxford and H. Raistrick, Biochem. Journ. 34 (1940), page 790; Photochemistry and Photobiology, Vol. 43, No. 6, pages 677- 680 (1986); Journal of Natural Products, Vol. 51, No. 6, Pages 1251/1256, Nov.-Dec. 1988; Photochemistry and Photo biology, Vol. 37, No. 3, pp. 313-319 (1983)).  

The extract for a pharmaceutical invention Zube Preparation can be obtained by harvesting freshly Coremi of mushrooms with an extractive agent.

The invention therefore also relates to a method for Preparation of an extract for a pharma according to the invention ceutical preparation, characterized in that one is fresh harvested mushrooms with an extractant ver rubs.  

Preferably, the extraction agents are glycols, glucose syrup, glycerine or liquid polyethylene glycol, in particular polyethylene glycol having a molecular weight from 200 to 400, and primarily with a Molekularge weight of 200 (PEG 200).

The process is preferably carried out by freshly harvested Koremia (conidia) in a porcelain rubbing peel or a porcelain ball mill with the extraction medium, preferably in a ratio of 5 to 10 ml / 100 mg of coremi. After the hearty rubbing (duration a few minutes) is the approach a few hours before preferably 8 to 10 hours, e.g. over night, in the air stand; the extract obtained is then, optionally after the decanting of deposited on the ground or filtrier th solids and / or dilution with water, preferably wise in the ratio 1: 1, subjected to sterile filtration. It can be worked at room temperature.

The production of mushroom cultures for the production of he According to the invention extracts and preparations can be used per se usual manner and using the usual nutrient media (See A. E. Oxford and H. Raistrick, Biochem. 34 (1940), page 790). However, it was different from the lite found that unhopped beer wort with 1.04 spec. Weight is a well-suited and inexpensive substrate. With such a breeding ground usually takes place from the very beginning fourth day a Koremienbildung.

Preferably, it is carried out at a temperature of 24 ° C, although other temperatures, e.g. one like that low temperature such as 16 ° C, are possible. The mushrooms are awake usually for 14 days, occasionally 6 to 10 Weeks, on the substrates. The mushroom hyphae and fruiting bodies are separated from the nutrient solution and can be used directly are processed.

The invention will be more apparent from the following examples explained without limiting it.

Examples example 1 Production of a Pharmaceutical Industry According to the Invention preparation from Penicilliopsis clavariaeformis Solms

Unhopped wort (d = 1.04) was used for the culture of this fungus. Temperature 22 to 25 ° C, growing time 1/2 to 2 months. The hyphae, coremi and fruiting bodies were taken out of the flask. The mushrooms can then be crushed well.

The freshly harvested bodies of Penicilliopsis clavariae Formis Solms were mixed with polyethylene glycol 200 (50-200 mg / 5 ml) in a porcelain mortar, the batch was left in the air overnight, then with diluted in distilled water (1/1) and sterile filtered filter (LR approach).

Example 2

The preparation prepared in Example 1 (LR approach) was together with reference samples for their inhibitory effect on HIV Viruses examined.

a) experimental procedure

For the experiments, 4 identical plates were used. Two of which serve for Profilerationsmessung on day 8 and day 10. The other two are for p24-Ag determination on day 8 and Day 10 (3 × 66 μl pooled).

b) Results

On the 8th and 10th day, the cells in all wells still looked like this  good, as with the controls (light microscope), except at the inventive approach LR (3.5 ug / ml), where a clear decreased cell count (approximately 25% compared to the control), taking the diminished cell could also notice the lower medium consumption, i.e. the color indicator in the medium still showed no acidification. Hypericin from a dosage of 0.5 ug / ml caused a German Liche p24 AG decrease.

Example 3

Comparison and study of the influence of synthetic Hypericin (HS + A), hypericin extract (HE ± A) with and without Albumin (A), albumin monomer without hypericin (A), fiction Approach (LR), Emodin (Em), Phosphatidylcholine and Phosphatidylcholine with hypericin (P and P-HS) on HIV-infe decorated lymphocytes.

Method: Lymphocytes from a normal donor (buffy coat-leucocyte-enriched blood) obtained from Ficoll gradients are incubated in two T-600 bottles in 75 ml Iscoves medium / 15% FCS 3.3 x 10 ⁵ Z / ml with 0.5% PHA and after a week incubation at 37 ° C, 5% CO ₂ infected with H9-III supernatant (one hour incubation for infection).

• 1. The thus obtained infected PHA blasts are clicked on the microtiter plates (4 pieces) (50 μl at 6.3 × 10 ⁵ Z / ml = 2.1 × 10 ⁵ Z / ml final concentration).
• 2. Add 50 μl of medium and
• 3. Clicked 50 μl of drug solution: HS + A 0.053; 0.375; 1.5; 2.25 μg / ml HE + A 0.375; 1.5 HE - A 0.375; 1.4 A same concentration as HS + A; 2.25 μg / ml LR 0.2; 1.0; 10 μg / ml em 9 μg / ml P 0.24; 0.6; 1.5 P-HS P 0.6 + HS 0.375; P 0.6 + HS 1.5

All solutions (1, 2, 3) are used for proliferation stimulation in the medium RPMI / 15% FCS 20 IU / ml TLF-IL2. Incubation at 37 ° C, 5% CO ₂ .

For proliferation measurement is incubated with radioactive ³ H-thymidine solution (1 ul ³ H-methyl-thymidine, Amersham) for 12 1/2 hours. The cells are then harvested on glass fiber mats and counted in the β counter ( b plate system).

p24-AG measurement with ELISA (Du Pont).

With regard to the HIV inhibition, the inventive Approach (LR) the best results.

Claims (20)

1. Use of anthraquinone or dianthrone dyes forming mushrooms for the preparation of pharmaceutical Zube TION. 2. Use according to claim 1 for the preparation of Zube preparations with an inhibiting effect on viruses and / or retroviruses. 3. Use according to claim 1 or 2, characterized that one uses mushrooms of the family Ascomyceten. 4. Use according to claim 3, characterized that Emodin or Scyrin-forming ascomycetes are used. 5. Use according to claim 3, characterized that Penicilliopsis clavariaeformis solms and / or his  Metabolism uses. 6. Pharmaceutical preparation, characterized that they contain extracts and / or ingredients of anthraquinone or dianthrone-dye-forming fungi such exists. 7. Preparation according to claim 6, characterized that they extracts and / or ingredients of mushrooms of the family Contains ascomycetes. 8. Preparation according to claim 7, characterized that they contain extracts and / or ingredients from Emodin or Scyrin containing forming Ascomyceten. 9. Preparation according to claim 7, characterized that they contain extracts and / or ingredients of Penicilliopsis clavariaeformis contains Solms. 10. Use of fungal dyes of anthraquinone or Dianthrone dyes forming fungi for the production of pharmaceutical preparations having an inhibitory effect on viruses and / or retroviruses. 11. Pharmaceutical preparation with an inhibitory effect on Viruses and / or retroviruses, characterized in that they one by triturating fungal dyes of anthraquinone and / or dianthrone type with glycols, glucose syrup, glycerol and / or liquid polyethylene glycol containing solution or consists of it. 12. Use of a preparation according to one of the claims 6 to 9 and 11 for the treatment and prophylaxis of virus diseases. 13. Use of a preparation according to one of the claims 6 to 9 and 11 to kill viruses and as viro static  Agent in vitro. 14. A process for the preparation of an extract for a Preparation according to one of claims 6 to 9, characterized gekenn draws that you can use freshly harvested bodies of mushrooms an extractant rubs. 15. The method according to claim 14, characterized that the extractant is one of the group Glycol, glucose syrup, glycerol and liquid polyethylene glycol. 16. The method according to claim 15, characterized that the extractant polyethylene glycol 200, 300 or 400 is. 17. The method according to any one of claims 14 to 16, characterized characterized in that the batch after rubbing some Hours in the air and the resulting extract, optionally after dilution with water, sterile filtered. 18. The method according to any one of claims 14 to 17, characterized characterized in that as a breeding ground for the fungal cultures Beer wort with a specific gravity of 1.04 used. 19. The pharmacologically described in the examples effective preparations. 20. The method of Her position of pharmacologically active preparations.