DE102004043463A1 - Differentiation of proteins, for use in diagnosis of cancer, comprises separating antigens from specimen electrophoretically and reacting with antibody composition - Google Patents

Abstract

Differentiation of proteins comprises: preparing an antibody composition, preparing a differentiation reagent; and preparing a support with wells for the antibody composition and a well for specimens. The antigens in the specimen are separated by electrophoretic treatment in the well and reacted with the antibody composition in the presence of the differentiation reagent. Independent claims are included for: (A) apparatus for differentiation of proteins using the method; (B) test kits for differentiation of proteins comprising a support as described, an antibody composition and a differentiation reagent; (C) pharmaceuticals for prevention and treatment of cancers and pre-cancers containing a fungal extract and optionally supports and fillers; (D) food supplements for prevention and treatment of cancers and pre-cancers containing a fungal extract and optionally supports and fillers, food supplements with immunomodulating effects containing a fungal extract and optionally supports and fillers; (E) use of fungal extracts to produce pharmaceuticals for prevention and treatment of cancers and pre-cancers; (F) use of fungal extracts to produce pharmaceuticals for targeted destruction of tissue, use of fungal extracts to produce pharmaceuticals with immunomodulating activity; (G) use of fungal extracts for prevention and treatment of cancers and pre-cancers; and (H) use of fungal extracts as described, in which the extract is produced by suspending fungi in a nutrient medium, disrupting their cell walls, incubating and filtration. ACTIVITY : Cytostatic; Immunomodulator; Antiangiogenic. MECHANISM OF ACTION : None given.

Description

The The present invention relates to a medicament for prevention and / or treatment of cancers and / or precancerous lesions, where cancer is a common one occurring, often deadly ending Disease is caused by a degenerated growth of malignant tumor cells the organism with uncontrollably proliferating and metastatic malignant Tumors penetrate and thereby destroy vital organs or parts of the organism. Precancerous lesions are morphological detectable precursors of malignant tumors.

Farther The present invention relates to drugs with immunomodulating Effect, on nutritional supplements for the prevention and / or cure of cancers and / or precancerous conditions or dietary supplements with immunomodulatory effect, as well as on the use of mold extracts for the preparation of such medicines or dietary supplements.

It are chemical agents, so-called cytostatics, known that the Inhibit cell growth and as a result of rapid disorderly growth malignant tumors these stronger attack as the healthy tissue. However, these cytostatic drugs often do not to a permanent cure of the cancer, but to one temporary containment and delay of tumor growth, allowing a successful use of cytotoxic drugs in the fight against cancer only limited possible is. Furthermore damage These cytostatic drugs due to their growth inhibition and the healthy Cells of the organism and in particular the immune system considerably, so their use is associated with serious side effects. moreover The use of cytotoxic drugs has been limited to certain types of tumors proved to be promising. Overall, so far none chemical agent for one permanently effective cancer control known.

It has therefore long been for alternatives to such chemical Wanted funds. The object underlying the present invention It was a natural one Agent for the prevention and / or treatment of cancer and / or precancerosis provide. This remedy should be as effective as possible as possible have low side effects. Furthermore, the agent should not Damage of the immune system, but if possible a beneficial effect to unfold on the immune system. It should preferably be both as Medicines can also be used as a dietary supplement. Another object was to produce as cheaply as possible for such To find means.

Mushrooms, Schitake, in particular, is becoming traditional in Far Eastern medicine for the prevention and / or cure of cancer as well as Strengthening used by the immune system. In contrast, there are molds in the Suspected due to mycotoxins produced by them, e.g. Aflatoxin to be carcinogenic. In the food industry will be Therefore, so-called noble mushroom cultures used. But also from the Mycotoxins produced by these noble mushroom cultures are becoming suspicious encountered.

In WO02 / 11563 becomes a dietary supplement described for the prevention and treatment of cancers, which is a biosynthesis product of Penicillium oxalicum var. Armeniaca CCM 8242 contains.

It was now surprising found that possible is to make extracts from molds that are in the form of medicines or nutritional supplements to the solution the above tasks can be used.

object The present invention is a medicament for the prevention and / or Treatment of cancers and / or precancerous lesions containing a mold extract and optionally pharmacologically acceptable carriers and / or fillers contains.

The medicaments of the invention is able to, due to its mitosis-inhibiting properties growth based on rapid cell division manifest Tumors to inhibit or counteract a further degeneration of precancerous lesions. In addition, it is suitable because of its proteolytic properties especially when administered locally, existing tumors and precancerous lesions dissolve. It is characterized by an anti-tumor effect. In addition, points it is a metastasis inhibitor especially when administered systemically and preventive effects. It is believed that beyond that also has an angiogenesis-inhibiting effect.

Although the extraction of the said mold extracts is in principle not restricted to certain molds, according to the invention molds of the genera Penicillium, Aspergillus and / or Fusarium and in particular molds of the species Penicillium candidum, Penicillium camemberti and / or Penicillium roqueforti are preferred. Suitable mold fungi strains can eg from the Wiesby GmbH & Co. KG, Niebüll, Germany (eg Penicillium candidum) or from the DSMZ - German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (eg Penicillium roqueforti and Penicillium camemberti).

Of the in the medicament of the invention contained mold extract is expedient a cell-free, preferably a sterile filtered and in particular a pyrogen-free extract.

Farther The mold extract is expediently an aqueous extract. alternative is also a dry extract and preferably a lyophilized extract possible.

Generally is the drug of the invention characterized in that the mold extract is a mold biosynthesis product, in particular contains a mycotoxin and / or a protease. In the context of the present Invention are mold biosynthetic products in synthetic form and natural To view mold biosynthesis products as equivalent.

The said medicines in contrast to the prior art chemical agents Immune system not. About that In addition, it is capable of the effect of this known chemical Synergistically enhancing agents or their harmful effects mitigate. In other words, at the same dosage of cytostatics significantly higher Efficiency or at lower dosage of the same efficiency be achieved. Thereby can The side effects of chemotherapy can be significantly reduced.

Consequently there is a preferred embodiment of the present invention in that the medicament of the invention additionally at least one per se known chemical agent for treatment of cancers and / or precancerous conditions, in particular a cytostatic agent which comprises, for example, cyclophosphamide, fluorouracil, endoxan, Cisplatin and mixtures thereof is selected.

Without to be bound by a certain theory is assumed that there is a so-called carrier effect here. That means, that the cytostatic is better introduced into the cells. As well It seems that the combination cytostatic / mold extract a much longer one Length of stay in the body has. In vitro studies of the mitotic rate in a cell culture have shown that the mitotic rate of the combined preparation significantly lower than the cytostatic alone. The same applies to the chromosome representation.

The said medicines So, as I said, in contrast to the chemical means of Prior art, the immune system is not. Preferably, it has an immunomodulating effect, based on its content of mold fungus extract based. Such a drug having immunomodulatory activity is therefore another object of the present invention represents.

One Another object of the present invention is a dietary supplement for the prevention and / or cure of cancers and / or precancerous diseases, which is characterized in that it is a mold extract according to the above Definition, optionally together with pharmacologically acceptable carriers and / or fillers, contains. Similarly, a dietary supplement with immunomodulating Effect, which is characterized in that it is a mold extract according to the above Definition, optionally together with pharmacologically acceptable carriers and / or fillers, contains another subject of the invention.

Further objects The present invention provides the uses of a mold extract according to the above Definition for the preparation of a medicament for the prevention and / or Treatment of cancers and / or precancerous conditions, for the production a drug with immunomodulatory activity, for the manufacture a dietary supplement for the prevention and / or cure of cancers and / or precancerous conditions and for the preparation of a dietary supplement with immunomodulating Effect.

in principle is the extraction of said mold extract not to certain Production process limited. However, it is advisable to choose a method in which a sufficient Effectiveness of the mold extract is guaranteed without the Extract then concentrated must become. Also, should the necessary cleaning measures expediently to a minimum. Furthermore, the mold extract, which is also proteolytic Has properties, does not decompose upon storage, what if necessary a sufficiently long maturation of the mold extract requires.

at the manufacturing process described below must all Steps to be performed under sterile conditions to a colonization to exclude with foreign germs. The use of a sterile workplace according to the current state The technique is therefore essential.

Here, a mold fungus lawn is first used in a suitable nutrient medium. This mold lawn is then suspended in an extraction medium. Subsequently, the cell walls of the mold fungus cells are broken. The resulting suspension is then optionally ripened, preferably using shear forces. Then the extract is filtered. The extract thus obtained is directly useful for the preparation of the above-described medicines and dietary supplements.

in principle is every nutrient medium suitable for the cultivation of mold fungi, provided it from the mold fungi contains usable carbon, nitrogen and energy sources. The Inoculation with molds is conveniently carried out by lyophilized mold spores in the selected one broth are given. Optionally, the mold can lawn later (once or more often) vaccinated on a new medium become.

Preferably the molds are first in a Sabouraud glucose broth and then on a Sabouraud glucose agar used for a mold mushroom lawn.

in principle any medium is suitable for suspension of mold fungus then the To break up mold cells, as long as it is ensured that the suspension medium the pharmacologically active ingredients of molds not destroyed. Preferably, an aqueous Medium used. Alternatively, e.g. an alcoholic or watery-alcoholic Medium usable. Particularly suitable is isotonic saline solution.

mold cells white very resistant cell walls on. The breaking up of the cell walls is therefore difficult. In principle, any method is suitable with the cell walls can be effectively broken if at the same time the pharmacologically active ingredients of Mold fungi are spared. According to the invention, the cell walls of the Mold fungus cells broken up in a cell disruption bomb. For this becomes the suspension of the mold fungus cells in the cell disruption bomb preferably repeated with nitrogen to a suitable pressure Of about 100 bar applied, each equilibrated for about 30 minutes and relaxed to atmospheric pressure, this cycle preferably performed at least ten times becomes. It is useful to break up the cell walls with ice cooling work.

The modalities The maturation of the cell suspension will vary from case to case and have to the individual case be matched. It has, however, been shown that a maturation of the suspension are carried out expediently can be done by keeping the suspension under sterile conditions for at least 8 months long daily is subjected to an ultrasonic treatment for one hour.

at the use of the said mold extract for the production a dietary supplement and in particular of a drug is of an adequate purity of the extract required. The extract is useful initially on glass wool and / or Celite prefiltered, then a filter with a maximum pore size 0.4 μm cell-free filtered and / or preferably over a filter with a maximum pore size 0.2 μm sterile filtered. For the manufacture of a medicament for internal use should the extract subsequently optionally be filtered pyrogen-free by ultrafiltration.

in the The invention will now be described by way of example and with reference on the attached Drawings closer explained. In the drawings show:

1 the course of the optical extinction of a mold extract in the range of about 300 nm to about 750 nm;

2 a photomicrograph of a blood culture smear without mold extract;

3 a photomicrograph of a blood culture smear with Penicillium roqueforti extract;

4 a photomicrograph of a biopsy sample of a type of Bowen's disease precancerous disease;

5 as 4 but after medical treatment with Penicillium candidum extract;

6 a detail enlargement of a chromosome representation for the determination of the mitotic rate;

7 a chromosome representation for the determination of mitotic rate in a blood culture without Penicillium candidum extract;

8th a chromosome representation for the determination of mitotic rate in a blood culture with Penicillium candidum extract;

9 a photomicrograph of a mouse liver cell culture with Penicillium candidum extract;

10 a photomicrograph of a mouse liver cell culture without Penicillium candidum extract.

example 1

manufacturing of a Penicillium candidum extract

The subsequent steps were taken at a sterile workplace laminar air flow performed. A Ampoule spore lyophilisate Penicillium candidum type SA (Wiesby GmbH & Co. KG, Niebüll, Germany) was in 500 ml Sabouraud glucose broth (Merck KGaA, Darmstadt, Germany) solved. About sterile filter and bubble application became the solution ventilated for two days. The resulting mycelium was cultivated on Sabouraud glucose agar (Merck KGaA, Darmstadt, Germany). After five Days had formed a continuous, about 3 mm thick mushroom turf, effortless removed with a sterile scalpel as a whole. 60 g of the fungal mycelium were in sterile conditions in 600 ml isotonic Saline given, with a stirrer suspended and then in a sterilized cell disruption bomb with 1850 ml internal volume (Model No. 4636, Parr Instrument Company, Moline, III., USA). The cell disruption bomb was under ice-cooling pressurized with nitrogen up to a pressure of about 100 bar ten times, Equilibrated for 30 minutes and relaxed to normal pressure. (Caution! For overpressure work are necessarily the relevant ones Safety regulations!) The suspension obtained was subsequently into a sterile Erlenmeyer flask, the air was mixed with inert gas (nitrogen) repressed, the flask was sealed with a stopper of sterile silicone rubber, eight Let stand for months at room temperature and daily one Hour of ultrasonic treatment in an ultrasonic bath subjected. Subsequently was first about glass wool, Celite and finally over one Filter with a pore size of 0.2 microns sterile filtered.

The extract thus obtained had the following properties: To determine the optical rotation, a polarimeter was filled with isotonic saline and the zero point was checked. The measurement of the extract showed a left turn of 0.3 °. The measurement of the dry residue of the extract gave a value of 1.08%. The measurement of the extract with a glass electrode gave a pH of 4.83. The results of the measurement of the optical extinction in the range from about 300 nm to about 750 nm (UV-Vis spectrum of the extract) are in 1 played.

wobei EA die Extinktion der Analyse, ES die Extinktion des Standards, EAB die Extinktion des Analysenblindwertes und ESB die Extinktion des Standardblindwertes ist. Es wurden folgende Konzentrationen errechnet: Probe mit isotonischer Kochsalzlösung: 10,4 g/100 ml. Probe mit Penicillium candidum-Extrakt: 8,8 g/100 ml. Demnach betrug die Eiweißreduktion in der Probe mit Penicillium candidum-Extrakt nach einer Stunde ca. 15 %.To measure the proteolytic activity of the resulting extract, calf serum (Newborn Bovine Serum, Flow Laboratories, Irvine, UK) was portioned into 2 1 ml aliquots. One sample was mixed with 1 ml of isotonic saline, the other with 1 ml of the extract. After a one-hour incubation period at room temperature (21 ° C), the total protein was determined in both samples. The measurement was carried out according to the generally accepted biuret method with a Zeiss spectrophotometer. Since the samples have a serum content of only 50%, the sample volume was doubled (200 μl). The calculation was made according to the formula:

where EA is the absorbance of the analysis, ES is the absorbance of the standard, EAB is the absorbance of the analytical blank and ESB is the absorbance of the standard blank. The following concentrations were calculated: Sample with isotonic saline solution: 10.4 g / 100 ml. Sample with Penicillium candidum extract: 8.8 g / 100 ml. Accordingly, the protein reduction in the sample with Penicillium candidum extract after one hour was approx 15%.

In Clinical self-testing was determined to be that concentration for injectives Applications of the extract was still too high; strong ones were formed Necrosis at the injection site, probably on the high Were due to protease content. After a dilution of the above extract in proportion 1: 3 with isotonic saline (corresponding to a protein reduction of about 5% in the diluted Extract) occurred practically after a subcutaneous injection of 1 ml no more necroses. Only a slight skin redness was so that this concentration can be considered practicable was.

In this diluted Penicillium candidum extract was L-γ-glutamyl transferase activity (measured with the monotest "γ-GT new" of Boehringer Mannheim GmbH, Mannheim, Germany; now Roche Diagnostics GmbH) 8 enzymatic Units per liter (8 units / liter). (See: Szasz, G., Persijn, J.P. et al., Clin. Chemistry and Clin. Biochem. 12 (1974) 228.)

For clinical Applications, however, require accurate standardization. It will be useful Cyclopiazonsäre standardized as Leittoxin which by HPLC chromatography (silica column, aqueous / alcoholic Eluent) can be determined.

Example 2

Proof of mitosis-inhibiting action of a Penicillium roqueforti extract in a blood culture

Venous blood was made inconspicuous with heparin solution heparin novo, 25,000 IU / 5 ml). The blood was centrifuged and the leukocytes, part of the Transferred 2 ml of plasma into 2 centrifuge tubes containing 7 ml of McCoy's 5A medium (Flow Laboratories, Irvine, UK). To a culture was added 70 mg of Penicillium roqueforti extract prepared analogously to Example 1 and 2 ml of isotonic saline. Only 2 ml of isotonic saline was added to the other culture. Both cultures were then incubated for 6 hours at 37 ° C. At the end of the incubation period, smears were prepared from both cultures and stained with Hemacolor (Merck KGaA, Darmstadt, Germany). In the untreated preparation ( 2 ) were counted on 100 leucocytes 63 cell divisions, in the treated preparation ( 3 ), there were only 3 cell divisions on 100 leucocytes. This experiment proves the mitosis-inhibiting effect of Penicillium roqueforti extract.

Example 3

Proof of mitosehemmenden effect of Penicillium candidum extract in one blood culture

In the same way was the extract of the mold Penicillium candidum according to example 1 tested for mitosis-inhibiting effect. The result was essentially identical to that of the mushroom extract Penicillium roqueforti.

Example 4

clinical Selbstversuch

The inventor suffered from Bowen's disease (precancerosis) on the right tibia. After securing the diagnosis by biopsy (cf. 4 ), the inventor treated himself by injecting daily 2 ml of the diluted Penicillium candidum extract according to Example 1 about 1 cm next to the diseased skin site. After two weeks of treatment the skin swelling had gone down and only one scar was visible. A repetition of the biopsy showed a completely unsuspected cell picture (cf. 5 ).

Example 5

chromosome representation (standardized method)

Two vials of Chromosome Medium 1A (Gibco, California, USA (now Invitrogen Corporation): unpublished mixture of Eagle's minimum essential medium, fetal calf serum, heparin, antibiotics, and phytohaemagglutinin) were each dissolved with 5 ml of the enclosed solvent. In one of the vials, 0.2 ml of the diluted Penicillium candidum extract according to Example 1, in the other, the same amount of isotonic saline solution was pipetted sterile. After both vials were warmed to 37 ° C, each 0.2 ml of venous blood was added. Thereafter, both samples were incubated at 37 ° C for 70 hours. At the end of the incubation period, 0.3 ml of colcemid solution (10 μg / ml) was added to each of the samples. After another two hours of incubation at 37 ° C, the cells were spun down and washed twice with hypotonic potassium chloride solution (0.07 M). Thereafter, the cells were dropped on a cooled slide and fixed. After staining with buffered Giemsalösung (pH 6.8) inclusion in Entellan ^® . Microscopic analysis showed a 70% lower mitotic rate in the sample treated with diluted Penicillium candidum extract according to Example 1. 6 shows a detail enlargement, 7 the blood culture without Penicillium candidum extract and 8th the blood culture with Penicillium candidum extract.

Example 6

Mitosis inhibition in cell culture the mouse liver

A freshly killed field mouse (Microtus arvalis) was removed from the liver under sterile conditions and minced with scissors. These liver particles were taken in a trypsinization flask with EDTA trypsin (Gibco, California, USA (now Invitrogen Corporation) and Eagle's Minimum Essential Medium.) This cell suspension was mixed homogeneously and divided into two equally large volumes ml of nutrient medium (MEM, 10% calf serum, 1% NEAA), after thorough mixing, this solution was filled into two 50 ml plastic culture bottles and one additional 5 μl of Penicillium candidum extract according to Example 1 was added to both incubated with an atmosphere of 20% O ₂ , 75% N ₂ and 5% CO ₂ at 37 ° C. The cultures were fed with new nutrient medium every 3 days, with new Penicillium candidum extract being added every three weeks. that cell division in the culture mixed with Penicillium candidum extract ( 9 ) was much slower than in the other culture ( 10 ).

Claims (24)

Medicaments for the prevention and / or treatment of cancers and / or precancerous conditions, characterized in that it contains a mold extract, optionally together with pharmacologically acceptable carriers and / or fillers. Medicament according to claim 1, characterized by an antitumoral, proteolytic, mitosis-inhibiting and / or metastasis-inhibiting Effect. Medicament according to claim 1 or 2, characterized in that the mold extract was obtained from a mold of the genera Penicillium, Aspergillus and / or Fusarium. Medicament according to Claim 3, characterized that the mold fungus the species Penicillium candidum, Penicillium camemberti and / or Penicillium roqueforti. Medicament according to one of Claims 1 to 4, characterized that the mold extract is a cell-free, preferably sterile filtered and in particular pyrogen-free extract. Medicament according to claim 5, characterized in that that the mold extract is an aqueous extract. Medicament according to claim 5, characterized in that that the mold extract is a dry extract, preferably a lyophilized extract. Medicament according to one of claims 1 to 7, characterized that the mold extract is a mold biosynthesis product, in particular a mycotoxin and / or a protease, if appropriate also in synthetic form. Medicament according to one of claims 1 to 8, characterized that in addition at least one per se known chemical agent for treatment of cancers and / or precancerous lesions, especially one Cytostatic, contains. Medicament according to Claim 9, characterized that the chemical agent consists of cyclophosphamide, fluorouracil, endoxan, Cisplatin and mixtures thereof is selected. Medicines with immunomodulatory effect, thereby characterized in that it is a mold extract according to the definition one of the claims 3 to 8, optionally together with pharmacologically acceptable carriers and / or fillers, contains. Dietary supplements for the prevention and / or cure of cancers and / or precancerous diseases, characterized in that it comprises a mold extract according to the definition one of the claims 3 to 8, optionally together with pharmacologically acceptable carriers and / or fillers, contains. Dietary supplements with immunomodulating action, characterized in that it has a Mold extract according to the definition one of the claims 3 to 8, optionally together with pharmacologically acceptable Straps and / or fillers, contains. Use of a mold extract according to the definition one of the claims 3 to 8 for the preparation of a medicament for the prevention and / or Treatment of cancers and / or precancerous conditions. Use of a mold extract according to the definition one of the claims 3 to 8 for the preparation of a medicament with immunomodulating Effect. Use of a mold extract according to the definition one of the claims 3 to 8 for the preparation of a dietary supplement for prevention and / or curing cancers and / or precancerous conditions. Use of a mold extract according to the definition one of the claims 3 to 8 for the preparation of a dietary supplement with immunomodulating Effect. Use according to one of claims 14 to 17, characterized that the mold extract was prepared by, under sterile conditions: a) in a suitable nutrient medium a mold mushroom was used, b) this mold fungus lawn was suspended in an extraction medium, c) the cell walls of the Mold fungus cells were broken up, d) the suspension obtained optionally, preferably using shear, a ripening was subjected, and e) the extract is subsequently filtered has been. Use according to claim 18, characterized that molds first in a Sabouraud glucose broth and then on a Sabouraud glucose agar were used for a mold mushroom lawn. Use according to claim 18 or 19, characterized that the mold lawn is suspended in isotonic saline has been. Use according to one of Claims 18 to 20, characterized that the cell walls of the Mold fungus cells were disrupted in a cell disruption bomb. Use according to claim 21, characterized in that the mold fungus cells in the cell disruption bomb were repeatedly charged with nitrogen to a pressure of about 100 bar, each equilibrated for about 30 minutes, were decompressed to normal pressure and that this cycle at least ten times. Use according to one of claims 18 to 22, characterized that the suspension obtained under sterile conditions at least Daily for 8 months was sonicated for one hour. Use according to one of Claims 18 to 23, characterized that extract first about glass wool and / or Celite was prefiltered, then a filter with a maximum pore size 0.4 μm cell-free was filtered, preferably over a filter with a maximum pore size 0.2 μm sterile was filtered, and then optionally filtered pyrogen-free by ultrafiltration.