ANTIMICROBIAL MEANS, PROCEDURES FOR THEIR PRODUCTION AND THEIR USE

The present invention relates to novel, as with antimicrobial activity [...] designated means microbial origin, process for their preparation and their use.

Bacteria and fungi produce a wide range of substances, together with the so-called secondary plants [...] shall be referred to as the. Many of these substances have importance, for example, as therapeutics and Feed additives. As producers of secondary metabolites have acquired in particular microorganisms scientific importance. The best-known representatives of secondary metabolites constitute the group, the antibiotics. Antibiotics are substances of microbial origin, the inhibit the growth of microorganisms even of small concentrations. One, it distinguishes between only substances which have an inhibitory effect (e.g. bacteriostats and antifungals) and callousing acting substances (such as bactericides and fungicides). Zur formation of mainly from the group of fungi and bacteria Antibiotics are from the group of actinomycetes capable [...].

The question, the interest for which they have at their respective site [...] -producing organisms, can not currently [...] & Nkulengu rail; end be answered. [...] is characteristic of that the metabolic pathways leading to them are not needed for growth and conservation of cells and enzymes. Based on the assumption that during the evolution has always only the Good stock, we have to consider in the antibiotics substances, the give a growth advantage in the areas where the producers, for example with regard to competitors is the same substrate,.

In State of the Art be au & Nkulengu rail; Several units also comprise so-called bacteriocins described. Here it is proteins, which can inhibit or kill and other bacterial strains of bacteria are formed in their growth. These substances are encoded by plasmids. Bacteriocins are isolated, for example, from E. coli ( [...] ).

A Particularity of antibiotics is that they inhibit the vegetative cells of other microorganisms, but not their spores. For the bacteriocins is characteristic that it can thus act only a narrow defined circle and inhibit relative specifically related microorganisms.

Sometimes also referred to as polypeptide-antibiotics acting Antimicrobially be polypeptides. Examples of this are and nicin subtilin. Nicin is from 29 to 34 amino acid residues is produced by streptococci constructed and. It has (120 °C) at elevated temperature in an acid medium (pH 2 to 3) inhibitory activity against spores of bacillus stearothermophilus, but even in those temperature condition, and is decomposed at neutral pH value. Nicin will take place, for example, use as a preservative for clostridia and [...] in cheese making. Subtilin similar properties as nicin and is produced by bacillus subtilis. Practical implementation has not yet found subtilin.

Total must be said that number and properties of the available antimicrobially active compounds is by far mattered not satisfactory. In particular, a need exists to antimicrobial agents, which act more effective, i.e. faster, elimination of bacterial spores allow a broad range of microorganisms and against.

It is the goal of the present invention provide novel antimicrobially active compounds, which are an effective controlling unwanted [...] on a broader basis. Means should be provided, in particular, which enable a more effective and more selective controlling microorganisms (provides & Nkulengu rail; t of vegetative cells and/or spores of these microorganisms).

This task has been solved by providing novel antimicrobially acting means Surprisingly, , which have are obtainable in a certain way and distinguish itself after cultivation of bacteria, under thermal conditions that they, in particular at elevated temperature, antimicrobial activity, i.e. vegetative cells of bacteria and/or bacterial spores and or vegetative not-bacterial cells, such as tumor cells, kill or inhibit [...] in a more efficient manner. Due to their characteristic antimicrobial properties at elevated temperature the [...] & Nkulengu rail shall be referred; en in the following also as " [...]" means.

It & Nkulengu rail; understands in an enlarged sense is antimicrobial activity. Microbes or microorganisms [...] in other words [...] & Nkulengu rail; bacteria, fungi, viruses, spores a higher eukaryotic systems but also cells, such as healthy or pathologically altered cells of the human or animal body.

A first article of the present invention relates to a method for producing a [...] & Nkulengu rail; en antimicrobially active agent, by thermal treatment after cultivation of a thermophilic a, mesophilic or [...], gram-positive or gram-negative, aerobic or anaerobic spore-forming micro-bacterium and washing the bacterial culture obtainable optionally washing phase.

A [...] & Nkulengu rail; useful, for the thermal treatment are obtained by washing a freshly made bacterial culture appropriate washing phase, such as a bacterial surface culture, with an aqueous washing liquid. Freshly cultured bacteria if possible quantitatively [...] culture medium from the separating the This and the cells treated in one or more from, preferably a, washing steps with the aqueous, preferably cooled, washing liquid. After each washing step the separation is carried out by filtration or preferably treated bacterial cells by centrifugation.

In any case contains the appropriate for the manufacture of an antimicrobial agent from the treatment of the culture with the one or more washing phase aqueous washing liquid and the resulting products separate ion step, which lead in the following thermal treatment on the innovative anti-microbial agents.

[...] & Nkulengu rail is a process for preparing a Preferably, ; en antimicrobially active agent, by

1. a) culturing a bacterium, preferably on a solid nutrient medium;
2. b) separating the bacterial culture from the nutrient medium with an aqueous washing liquid and washing the bacterial culture;
3. c) separating the resulting liquid washing phase of the bacteria; and
4. d) thermal treatment of the washing liquid, preferably at a temperature of at least about 120 °C, preferably at a pressure of at least about 1 atmosphere and preferably over a period of at least about 5 min.

[...] & Nkulengu rail Implementation of the; en production method is not limited to certain bacterial genera. It & Nkulengu rail; it was found, surprisingly, in other words, since & Nkulengu rail; substantially all genera of bacteria independently of the respective morphological or physiological properties useful represent [...] -sources. Even the respective degree of pathogenicity of the bacterium [...] & Nkulengu rail is; not relevant. For the purposes of the further description of the invention are distinguished in terms of their request to the growth temperature only the respectively examined bacteria. It & Nkulengu rail; insertable [...] bacteria have an optimum of growth (provides & Nkulengu rail; t a maximum growth rate) below about 20 °C. It & Nkulengu rail; useful mesophilic bacteria have an optimum of growth at a temperature in the range of about 20 to about 40 °C. It & Nkulengu rail; useful thermophilic bacteria have an optimum of growth above about 40 °C, such as at about 40 to 110 °C. Under above definition usable bacteria shall also be covered by such bacteria, as optional thermophilic, optionally are mesophilic or optionally be State of the Art [...] in the described. The [...] & Nkulengu rail; which can be inserted are bacteria au & Nkulengu rail; Several units also comprise irrespective of their ability to sporulation useful.

Non limiting examples of spore screen end bacteria are selected under B. stearothermophilus, B. subtilis, B. licheniformis and B. cereus. Non limiting examples of [...] & Nkulengu rail; not- spore screen end bacteria selected from E. coli are viable, Y. pseudotuberculosis, Y. pestis, m. [...], p. aureus and E. faecalis.

For the cultivation is preferably used a solid nutrient medium, the optimum growth of the respective bacterium necessary nutrients and for which the trace elements have been added. It & Nkulengu rail; useful solid nutrient media are agar-media, which agar at a concentration of about 15 to 20 grams per litre. The preparation of suitable solid approaching media is familiar with some of the person skilled in the art. The culturing is carried out under the respective optimum growth conditions depending on the bacterium (temperature, atmosphere, brightness, pH value, etc.). Particularly is preferably the production of surface cultures with said device on a flat, solid nutrient medium, such as solid agar plates.

The [...] & Nkulengu rail; e manufacturing method, however is not limited to a cultivation on solid media. The cultivation of the bacteria in semi-solid or, in principle, also suitable Rather, liquid culture media. Also these culture techniques and methods for the isolation of bacteria contained therein are familiar with some of the person skilled in the art.

In a next process step the [...] is separated & Nkulengu rail; e bacterial culture from the nutrient medium or from the culture broth, preferably during the late logarithmic growth phase or during the early stationary growth phase. Is washed with an aqueous washing liquid after the separation. As preferred [...] & Nkulengu rail is used washing liquid; sterilised, preferably distilled water or physiological saline solution. Sterilization or disinfection of the washing liquid can take place by, for example, sterile filtration The.

This washing step can be carried out one or more times. Culture is brought into contact with the washing liquid thereto. The washing liquid is then separated, optionally after brief incubation, but preferably without further incubation of the bacteria again. This is done by filtration or centrifugation. Centrifugation is preferably. Preferably, 2000 (RCB) is carried out at a relative centrifugal acceleration [...] from about 30000 g to g, 2500 g to about 25000 g such B (calculated from the formula: RCB = 1.119*10^{-5 *} rpm^{2 * r}; wherein rpms for the number of revolutions per minute and r is the rotor radius is). Preferably, cooled 4 °C e.g. at during centrifugation. 5 min to 2 hours is e.g. [...] The. 30 minutes is conducted at, for example, the centrifugation (3000 rpm at a rotor radius of 25 cm corresponding to) 2500g with or without cooling. It is important, since & Nkulengu rail; the bacterial cells in this washing step not chap, so since & Nkulengu rail; Cell Value emerges in the washing liquid.

The thus obtained cell-free supernatant (i.e. the washing phase) is subjected to a thermal treatment in a further step. Preferred treatment conditions are:

1. i) temperature: about 100 to 200 °C, such as 110 to 180 °C, in particular about 120 °C;
2. ii) pressure: about 1 to 3 atmospheres, 2 atmospheres in particular about 1.5 to;
3. iii) duration: 1 minute to 1 hour approximately, in particular approximately 5 minutes to 30 minutes, preferably about 5 to 10 minutes.

The optimum parameter combination for the above temperature treatment can be established by means of less attempts. The temperature treatment can be carried out, for example, by autoclaving in conventional autopiano devices.

The in this way by thermal treatment of the washing phase [...] & Nkulengu rail available product can as such to the; en purposes are used.

However, there is au & Nkulengu rail; Several units also comprise the possibility, the further limit thermal bio tables activity in the washing phase. This fitting is, for example, a size & Nkulengu rail; [...] by. Suitable size & Nkulengu rail; [...] are known to the person skilled in the art. For example, there is the possibility the size & Nkulengu rail; [...] carry out by means of column chromatography. Suitable also requires, however, the size & Nkulengu rail; [...] by greater & Nkulengu rail; [...] filtration. For example, a selective filtration with filter of a [...] & Nkulengu rail; e of about 0.22 µm be carried out. One receives a processed here as a filtrate [...] -Preparation components thereof a size & Nkulengu rail; e in the range of less than about 500 daltons. The or have a molecular weight in the range of this preparation the antimicrobially active ingredients about 60 to 450 Daltons, preferably about 60 to 120 or 80 to 200 or 100 to 300 Daltons, such as 200 to 300 Daltons. Preferably, determining the molecular weight by means of thin layer chromatography (Stationary phase: silica gel, Fa. [...], the Czech Republic; Mobile phase: ethyl alcohol 95% strength aqueous concentrated; with urea (about 300 Daltons) (about 60 daltons) and Penicillin as standards). Another containment of the antimicrobially active ingredient (the antimicrobially active components) would be possible with conventional preparative methods is not necessary, however, for practical reasons.

The [...] & Nkulengu rail; obtainable thermally treated, and optionally greater & Nkulengu rail; [...] washing phase is without loss of activity at about 2 to 6 months and longer and when dried in the liquid 8 °C 36 months and-critical storage stable longer.

[...] & Nkulengu rail; a further variant of the [...] & Nkulengu rail; en manufacturing process is the thermally treated and optionally greater concentrated & Nkulengu rail; [...] washing phase to dryness. As has proved to be particularly suited is the drying of the liquid phase at 37 °C for this purpose, normal pressure and over a period of several hours to several days. Other drying techniques, such as the freeze-drying would also be useful. A thus realized can dry preparation in water before use or other suitable aqueous carrier, such as a physiological saline or an injection solution, residuelessly Resolving.

Another the subject of the present invention relates to the, for example, according to one of the above-described [...] & Nkulengu rail; en Method obtainable, antimicrobially active and [...] designated as means. This are in particular characterized in that their active ingredient (active ingredients) has a molecular weight in the range of less than about 500 Daltons, such as about 60 to 450 Daltons, such as 70 to 400 Daltons, especially about 100 to 300 Daltons, more preferably about 200 to 300 Daltons (have).

In operational point of the are antimicrobially active means the present invention characterized in that they have at least an antimicrobial activity, the is chosen from:

1. a) inhibitory action on a heterologous and/or homologous bacterial spores; and
2. b) homologous vegetative bacterial inhibitory action heterologous and/or and/or vegetative not-bacterial cells;

hetero log in this context means, since & Nkulengu rail; the [...] from another bacterial species was isolated from the bacteria or [...]spore species than the inhibited.

[...] eukaryotic organisms vegetative one cells are cells one-or multi-cell, by mitosis are capable to cell division.

[...] cells comprise normal and in particular peccant modified cells, such as, in particular, tumor cells.

A [...] & Nkulengu rail; e thermally controlled activity is given, when the anti-microbial effect is inducible by increasing the temperature. In particular, the temperature increase is effected to a value, the lies above the physiological temperature optimum of the target cells or spores to inhibiting.

In particular are & Nkulengu rail [...] ; en-which comprises means preferably that it (such as cells or somatic cells or tumor cells [...] ) vegetative cells inhibit, when the temperature is adjusted to a value in a range, whose lower limit temperature T_{u about} the highest temperature corresponds to, bie which the still are capable of continued propagation and its upper limit temperature T_{o vegetative cells to be destroyed approximately} corresponds to the highest temperature, in which these cells are still viable but no longer capable of continued propagation.

It & Nkulengu rail; vegetative cells are specified in the following typical temperature ranges for the inhibition, without being reduced, however, point:

• vegetative cells [...] bacteria: T_{u =} 10 °C T_{o =} 45 °C, such as 20 to 40 °C;
• vegetative cells [...] bacteria: T_{u =} 40 °C T_{o =} 70 °C, such as 52 °C
• vegetative cells of thermophilic bacteria: T_{u =} 70 °C T_{o =} 120 °C, such as 75 to 100 °C.
• Tumor cells: T_{u =} 41 °C T_{o =} 54 °C, such as 43 to 48 °C, or 44 to 46 °C or 43.5 °C.

It & Nkulengu rail; e antimicrobially active agent with spores-inhibiting activity need advantageously in order to extend their spores-inhibiting effect a temperature in the range of about 100 to 150 °C, preferably 110 to 130 °C, in particular about 120 °C.

Wise Men [...] & Nkulengu rail the; en means a bacterial spores inhibitory effect on, so this is in a characteristic manner over a wide pH range, especially over a pH range of 2 to 9, preferably about 6 to 8, in particular 7 to 7,5, unlike the known and described above from the detectably nicin State of the Art.

Vegetative bacterial cells is typically observed in an inhibitory effect on The pH range of about 4 to 9.

The duration of the treatment is [...] & Nkulengu rail; depending on the temperature selection is performed as well as upon the concentration of spores or vegetative cells to inhibitory [...] as well as upon the concentration of the. Typically, the duration is 1 minute to 5 hours, however, in a range of the temperature treatment, preferably in a range from about 5 minutes to 1 hour, such as 5 to 15 minutes. The temperature treatment of tumor cells (by, preferably local, hyperthermia) 20 minutes to 4 hours duration usually, such as 2.5 hours, one-or several times, such as 2bis 4-times per week.

A [...] & Nkulengu rail; it antimicrobially effective [...] & Nkulengu rail [...] is located in a; en Preparation then before, when attached as spores of bacillus stearothermophilus and/or vegetative cells of activity characteristic at least and/or vegetative cells of Escherichia coli inhibited Bacillus stearothermophilus.

The [...] & Nkulengu rail; e effect is, as explained by the later described embodiments is even more precise, to a particular microorganism or not. Spores of a particular microorganism limited. Activity patterns of a plurality of differentially more pronounced reword [...] & Nkulengu rail Instead; en means. Allen means is common and their thermal stability, however, whose significant activity development at elevated temperature.

The [...] & Nkulengu rail use of the; en [...] in a variety of different fields of medicine and of industrial technology should take on no serious obstacles. We have studied to date have been obtained from non-pathogenic microorganisms [...] Most of the, called a good storage stability showed no pathological changes and for mice. Products obtained from pathogens [...] no pathogenic effects the Even. A pharmacological application of [...] & Nkulengu rail; en products is therefore appropriate.

The subject of the present invention are therefore au & Nkulengu rail; Several units also comprise pharmaceutical compositions, which in a pharmaceutically acceptable, an effective amount of a solid or liquid carrier [...] effective means according to & Nkulengu rail; contain defined above.

The administration of the [...] & Nkulengu rail; en means is not limited to certain procedures. As Examples of suitable routes of administration can be mentioned: intraarterially, intravenously (e.g. by infusion), intramuscularly, subcutaneously [...], orally, nasally, buccally, sublingually, retrobulbar, per [...], vaginally, rectally, [...], dermally, intradermally, peritoneal, [...], intratumorally, electrophoretically, than depot implant, via catheter, as well as administration in eye and ear.

The for a particular pharmacological application dose to be administered is a function of several factors, such as from the specific antimicrobial activity of the respective [...] -preparation, which can be strong origin organism different depending on.

Also the dosage is [...] & Nkulengu rail; t of the nature of the bacterium to be combated, and the general condition of the patient to be treated by the severity of the disease.

Because contraindications for the [...] & Nkulengu rail; en [...] as well as side effects of the preparations are not known, the therapeutic dose to be administered on the basis of clinical effect may and the ability of the patient from the organism which excrete [...], are determined. There is also no contraindications for the combined application of [...][...] and antibiotics as well as and chemotherapeutic agents, and in particular of such chemotherapeutic agents, the promote secondary infectionsimmune forces of the organism and thus weaken the generation of the.

Generally should lie in the range of about, however, a suitable therapeutic dose of 5 to 800 micrograms per kilogram of body weight [...] -preparation.

The [...] & Nkulengu rail; en [...] can be used individually or in combination with other pharmacologically active substances. Examples of particularly suitable active substances are [...] : nifedipine, plasmin, plasminogen, beta-blockers, acetylsalicylic acid, prednisolone, phenobarbital, paracetamol- [...], EDTA, heparin, quinine, atropine, theophylline, corticosteroids any kind, anticonvulsants of any kind, digitalis, verapamil, antidepressants any kind, antibiotics any kind, antiepileptics, central analgesics, [...], antiparkinson agent, antihistamines, tricyclic antidepressants, diuretics, antidiabetics, muscle relaxants of all kinds, Aminoglutethimide, dexamethasone, digitoxin, asparaginase, medroxyprogesterone acetate, megestrol acetate, ace inhibitor, azathioprine, allopurinol, etoposide, cyclophosphamide, cyclosporin A, lipopolysaccharides any kind, doxorubicin, daunorubicin, diclofenac, [...], famotidine, fluconazole, [...], [...], griseofulvin, indomethacin, [...], josamycin, ketoconazole, [...], mesna, melphalan, antifolate, methotrexate, morphines and opiates any kind, omeprazole, rifampicin, sulfadiazine, pentostatin, pethidine, thymidine, procarbazine, ranitidine, hydroxyurea, tamoxifen, idarubicin, Amphotericin B, [...], chlorambucil, immune materials of all kinds, interleukins of all kinds, immunotoxins, ricin- [...], diphtheria toxin, interferon-alpha, beta and gamma, estrogens, phenazone, warfarin, [...], navelbine, Let and selenium containing compounds, [...], thiols, G-SH-acetyl-G-SH, trimethoprim, [...], vinblastine, vincristine, cimetidine, [...], [...], mitoxantrone, estramustine phosphate, thiotepa, procarbazine, nitrosoureas, CCNU, ME-CCNU, BCNU, [...], [...], UM -26, VP -16, Hexitol derivatives, DAG, [...], DIAC, butyl-scopolamine, baclofen, opioids, [...], tramadol, [...], codeine, clonazepam, [...], clonidine, morphine, hydromorphone, L- [...], L-methadone, fentanyl, [...], haloperidol, [...], Metamizol, cytarabine, dapsone, bleomycin, acetyldigoxin, cisplatin, carboplatin, platinum saltsplatinum preparations and any kind, phenytoin, oxygen, nitrous oxide, busulfan, [...], ifosfamide, sodium thiosulfate, carmustine, old type [...][...] -Vaccines and vaccines, [...], mitomycin c, [...], [...], plasma proteins, salicylates, levamisole, pyridoxine, [...], fluorouracil, [...], [...], cysteine, [...], [...], hexamethylmelamine, lomustine, semustine, mercaptopurine, [...], Nucleosides and nucleoside analogs of each type, alcohol, aminophenazone, antirheumatics, glafenine, ketoprofen, phenylbutazone, prednisone, probenecid, mitomycin, Pharmaceutic, calcitonin, glucagon, furosemide, [...], mitoxantrone, [...], [...], paclitaxel, taxol, quinidine, [...], vitamins A, B, carbon, D and E, antioxidants of all kinds, each kind blood coloring materials, procarbazine, [...], lithium, phenytoin, toremifene, [...], [...], [...], [...], fluorine arabine phosphates, [...], midazolam, sugar solution, [...], [...], pindolol, [...], [...], sotalol, magnesium, spironolactone, triamterene, aminoglycosides, DNA-RNA extracts from Stöhr and calf thymus, betulinic acid, [...][...], glutathione (GSH) and GSH-derivatives.

Appropriate [...] & Nkulengu rail dosage forms; he [...] comprise e.g. aqueous solutions, aqueous infusion, [...], ointments, creams, sustained-release preparations, such as implants or Plaster containing an active substance and the like.

The subject matter of the invention are au & Nkulengu rail; Several units also comprise disinfectant solutions, comprising, in a usual for disinfecting suitable, liquid, inert carrier a antimicrobially effective amount of a at least agent according to & Nkulengu rail; defined above. [...] can & Nkulengu rail If desired, the; en means in combination with other already known and already in use for the purpose of disinfection State of the Art from the substances are used. Au & Nkulengu rail; Several units also comprise it could conceivably, combinations [...] & Nkulengu rail; it comprises means of different origins ( [...] /are mesophilic, [...] /thermophilic, or are mesophilic/thermophilic) to use.

The is a function of several factors to be used for disinfection dose, such as from the specific antimicrobial activity of the respective [...] -preparation, which can be strong origin organism different depending on. Also the dosage is [...] & Nkulengu rail; t of the type of bacterium, and of the seriousness of the contamination to be combated. Generally should, however, a suitable disinfectant dose lie in the range of about 10 to 100 mg per litre disinfecting solution [...] -preparation.

For the purpose of preserving food [...] & Nkulengu rail should the; en preparations in a dose of approximately 1 to 8 mg/L food products to be used.

Another subject of the invention relates to active compound combinations, comprising at least one [...] & Nkulengu rail; and at least one surface-active [...] it, preferably [...] substance; and their use to the purposes described herein, in particular as antibacterial agents. It was observed in other words [...] & Nkulengu rail; surprisingly, since & Nkulengu rail; such combinations of substances have a significantly enhanced antimicrobial effect. In principle all customary surfactants are useful, as long as it does not adversely affect the effectiveness of the [...]. [...] Examples of suitable surface-active substances are ammonium salts of the general formula R (R²⁾_{3 + X-}1 N^, wherein R^{1 for a} long chain, linear or branched hydrocarbon group to 25 carbon atoms with too, the radicals R^{2 are identical or different}, represent a hydrogen atom or a hydrocarbon radical having up to 4 carbon atoms, which are optionally with 1 stands are, 2,3 or 4 hydroxyl groups may be substituted, , or two of the radicals R together with the^{nitrogen atom to which they are attached for a} 2 4bis 7-membered, optionally aromatic, heterocyclic ring, and X is a conventional counterion, especially a halide ion, such as Cl or Br,. Examples_8-20- are in particular mono-C [...] orC_8- carbon_20- carbon alkyl pyridinium salts, such as cetylpyridinium chloride. The surface-active agent in a proportion of 0 [...] -preparation in the can, 001 to 0.1, preferably about 0,0.05 wt. - % be contained 005 to. Neat or in a dilution of 1:10 can [...] -Preparation The 1:1000 in customary diluents to, such as water, are mixed with the surfactant.

Therefore, we have, for example, be established, since & Nkulengu rail; a [...] & Nkulengu rail; it from a 1:1 mixture (cells) from Bacillus thermophilus or [...] Bacillus stearothermophilus and 100-fold dilution of and addition of 0.01 E. coli according to 10 wt % of cetyl pyridinium chloride or^{6 B.} stearothermophilus-spores. E. coli-spores over a period of 20 to 30 min at 75 °C kills already. Among the same experimental conditions (temperature, time, concentration) could for the individual components are detected ( [...] or surfactant) is no comparable antibacterial effect.

The subject matter of the invention is also the use of a [...] & Nkulengu rail; en [...] for treating or preventing local or systemic bacterial infections in humans or animals. As examples can be referred to as a use for the treatment of burns, red sound, dermatitis, urogenital infections, chronic infections of internal organs, infections of the nasal pharynx, the eye sockets or of the inner ear, and for treating secondary infections ; particularly preferably in combination with a local or systemic hyperthermia-treatment [...].

The subject matter of the invention is also the use [...] & Nkulengu rail; he [...] or means for disinfection or sterilization of food or medical devices or for the preservation of foodstuffs.

Particularly [...] & Nkulengu rail; ig [...] is & Nkulengu rail the use; he [...] in the production of the sensitive food products, the require heat sterilizing, the quality of the foodstuff further increased and a shortening of the exposure period.

[...] & Nkulengu rail A preferred embodiment concerns the use; it comprises means to pharmacological purposes in combination with another form of therapy, selected from chemotherapy, radiation therapy and hyperthermia.

In combination with a local or general [...] the [...] can be used for the treatment of acute and chronic contagious diseases, such as prostatitis, [...], [...] or infections, such as pneumonia.

A large area for application [...] & Nkulengu rail; he [...] umfa & Nkulengu rail; t the treatment of primary and secondary immunodeficiency-states, e.g. at AIDS and similar disease conditions, as well as in tumor therapy with cytostatics, where often threatening bacterial [...] occur.

Au & Nkulengu rail; Several units also comprise, surprisingly, it was found, since & Nkulengu rail; the [...] & Nkulengu rail; en an inhibitory activity against neoplasms also have antimicrobial agent. This inhibiting effect manifests itself in a suppression of tumor growth and/or already formed tumors [...][...] a. This effect is observed when administered [...] & Nkulengu rail particular; he [...] under hyperthermic conditions. In order to further increase the anti-tumor activity can be carried out together with conventional anti-tumor active substances[...] a administration of the, which reduce the resistance of the tumor cells. Examples of suitable active substances are mentioned for combining 5-can As fluorouracil, [...], [...], [...], methylhydrazine, [...], procarbazine and dacarbazine.

The subject matter of the invention is therefore au & Nkulengu rail; Several units also comprise a use [...] & Nkulengu rail; it includes means for the treatment of solid or not-solid, high or low differentiated tumors and [...][...] of various origins, in particular in combination with hyperthermia.

radiationtherapeutic Ma & Nkulengu rail; took, as the irradiation with gamma rays, laser beams or ultraviolet light with a [...] & Nkulengu rail can be; en [...] -therapy combine well, because these physical factors [...] reduce the resistance of the microorganisms against the.

Because [...] -therapy by raising body temperature the effectiveness of can be increased is also the simultaneous or time advanced administration more fever-promoting means, such as, e.g. from [...], polysaccharides and other substances, [...] & Nkulengu rail; ig. A accompanying hyperthermal treatment is particularly preferably.

Because the [...] & Nkulengu rail; en [...] (about 2 to 10 hours) from the organism are excreted rather quickly, the application is especially in chronic diseases [...]more depositing[...] -preparations at the same number displayed.

To should be determined to optimize the effectiveness a [...] -therapy, preferably before the response of the pathogenic microflora [...] a treatment on the first. These facilitates the choice of the optimum [...] -preparation. For [...]spore screen end Bacteria should e.g. such are used, the inhibit not only the vegetative cells, but also their spores.

Taking [...] & Nkulengu rail the ability of the; en [...] at elevated temperature to remove tumors, it is necessary, to examine their activity against various neoplasms, in particular in combination with other physical and chemical auxiliaries, which can weaken the resistance of the tumor cells.

For the [...] -Application condition for effectiveness (local or systemic) raising body temperature is the. The level of temperature increase and the exposure period point of application[...] depend on the properties of the as from the, and thereof, as the patient sits uncomfortably the elevated temperature. Methods and devices for hyperthermic treatment of the human or animal body are known to the person skilled in the art.

The invention will now be described on the basis of the following further explained not-limiting examples.

Three methods have been mainly used On reading the [...] -activity.

In glass or plastic containers were presented at equal volumes and spores [...] (0.01+0.01 ml). In any assay mixture (e.g. 10⁶⁾ a was accommodated spore quantity Total identical. 24 hours at 37 °C is weathered The containers. The bio test were inserted in special bag prior to autoclaving. The autopiano duration was mostly 0, 5, 10, 15, 10, 25 and 30 minutes at the same temperature (starting of exhaustion of air from the sterilization chamber). autopiano duration is at least used for each 5 biological testing. The following control tests also were verified: 1) bio test, the not have been treated in the autoclave; 2) bio test from a spores-water-mixture (as "water" the flushing water from the served [...]agar media ); 3) (1.5 µl) container with nutrient medium. 0.5 ml was added for detecting viable spores after the autoclaving each biotest a liquid indicator-containing nutrient medium of the following composition:

The spore speciesbio test were then incubated at the optimum temperature, depending on the tested. Useful for the detection of spores of the 48-hour incubation at 55 °C [...] B. stearothermophilus was carried out, for example, a. Viable spores in a biotest before layers, so is a colour change to yellow observed.

The mathematical evaluation of the results obtained from the total number (A) was carried out by determining the activity index (colour change to yellow) in the positive results (P) and wherein the control beginningstest beginnings on the basis of the formula (Q) [...] -containing: ((P-Q)/ P)* 100 = A

test beginningsmany control and always have been reviewed.

Analog proceeded for tests of the same type is, but which have been carried out with vegetative cells instead of spores.

Here different amounts were mixed ( vegetative one cells or spores) with [...] of microbes. After concentrating to dryness the bio test have been exposed to an elevated temperature. [...] & Nkulengu rail; end the lugs were offset (e.g. 55 °C) cultured with the above nutrient medium and at suitable temperature. A colour change of the indicator according to the presence of spores showed yellow.

In glass or plastic containers was added a mixture of same volumes [...] Here Spore Suspension (^{5 spores} per approach 10) and. The [...] were diluted to different extents. 24 hours at 37 °C Man dehumidified container contents. Approximately 15 minutes were autoclaved at 120 °C bio test The.

After addition of equal amounts of the described above under suitable conditions the lugs were cultured nutrient medium. A colour change of the indicator showed the presence of spores on.

A culture of thermophilic bacterium B. stearothermophilus, strain [...] B -718, has been sown on solid [...] -agar medium (1% in weight [...], 1.5 wt. - % agar, 0.5 wt- % NaCl, pH 7.2 ± 0.1,20 min at 121 °C autoclaved; 9 cm diameter with agar bowls ). The agar culturescultural cabinet at 55 °C 18 hours were inoculated in a maintained. The was rinsed with sterile microbial yield, (+ 4 °C) distilled water cooled (3 ml per agar-shell) from the solid nutrient medium in a centrifuge tube. 30 minutes at 3000 rpm was centrifuged microbe suspension obtained Precisely with. 5 minutes at 120 °C The supernatant was then autoclaved. The solids content of the preparation of 37 °C 12 mg/ml. in drying amounted to

Zur pipetted in 0.02 ml of a mixture of determining the activity quantity of the above each Eppendorf, -Glasses [...] obtained at different dilution and a same from B. stearothermophilus (^{6 spores} 10) Spore Suspension. 10 identical approaches have been prepared from each dilution. 24 hours at 37 °C test beginnings Keep prepared were dried. The [...][...] -preparation contained distilled water in place of the control tests were dried and manner, as the test beginnings.

The 10 minutes at 120 °C bio test were cooled to room temperature and autoclaved. In were added 0.5 ml of the above liquid thereafter the Eppendorf, -glasses per 48 hours at 55 °C and the samples were indicator-containing nutrient medium thermostated. The tests were evaluated discoloration of the nutrient medium Compliance. One received the following results:

For the monitoring results, follows, since & Nkulengu rail; the without dilution was active [...] -preparation of example stearothermophilus, 1:10000 dilution was inactive at 1:100 [...] and wherein its activity decreased.

The significantly faster removal of spores by autoclaving [...] & Nkulengu rail in the presence of the; en [...] is illustrated by the following test results. It has been applied above method 1:

A culture of the mesophilic bacterium Bacillus cereus was sown on solid [...] -agar medium (1% in weight [...], 1.5 wt. - % agar, 0.5 wt- % NaCl, pH 7.2 ± 0.1,20 min at 121 °C autoclaved; 9 cm diameter with agar bowl ). The agar culturescultural cabinet at 37 °C 18 hours were inoculated in a maintained. Yield was rinsed with cooled distilled water microbial The (3 ml per agar-shell) completely in a centrifuge tube. 30 minutes at 3000 rpm was centrifuged microbe suspension obtained Precisely with. 5 minutes at 120 °C The supernatant was then autoclaved. The 37 °C 11 mg/ml. [...] after drying was in

Zur pipetted in 0.02 ml of a mixture of determining the activity quantity of the above each Eppendorf, -Glasses [...] obtained at different dilution and a same from B. stearothermophilus (^{6 spores} 10) Spore Suspension. 10 identical approaches For each dilution is used. 24 hours at 37 °C test beginnings Keep prepared were dried. The control tests were contained in the same manner instead of the distilled water and dried [...] -preparation test beginnings as the.

The 10 minutes at 120 °C bio test were cooled to room temperature and autoclaved. In were added 0.5 ml of the above per thereafter the Eppendorf, -glasses and the samples were 48 hours at 55 °C indicator-containing nutrient medium thermostated. The tests were evaluated discoloration of the nutrient medium Compliance.

It became obvious, since & Nkulengu rail; [...] -preparation of example 1 up to the dilution of the cereus: 10000 (maximum tested dilution) (activity index: 100%) was effectively. Without [...] were not effective with respect to the spores 55 °C the temperature increase to. Control tests, have been used in which only spores of B. stearothermophilus, after heating at 55 °C viable microbes contained. In tests with no viable bacteria spores and [...] developed after the heating.

A [...] Yersinia pseudotuberculosis was sown [...] -agar medium on solid culture of (1 wt. - % [...], 1.5 wt. - % agar, 0.5 wt- % NaCl, pH 7.2 ± 0.1,20 min at 121 °C autoclaved; 9 cm diameter with agar bowl ). The agar culturescultural cabinet 10 °C 96 hours were inoculated in a held at. Yield was rinsed with cooled distilled water microbial The (2 ml per agar-shell) completely in a centrifuge tube. 40 minutes at 3000 rpm was centrifuged microbe suspension obtained Precisely with. 5 minutes at 120 °C The supernatant was then autoclaved. The 37 °C 10 mg/ml of the preparation was after the drying, to solids content.

Zur determining the activity is carried out in 0.1 ml of the 1 ml pipetted each sample tubes 18-hour culture of [...] -preparation and a (2*10^{4 microbial cells} per milliliter) mixed E. coli. The vial lie & Nkulengu rail; 52 °C are of different lengths in a bath of water at is. As controls an approach is used, in which a mixture of the water had been added in place of the and [...][...] and bacteria, the tested [...] without heating. On completion of each exposure was sown an aliquot (0.1 ml) in the thermostat 72 hours on a agar-plate and each mixture incubated at 37 °C. [...] & Nkulengu rail; end the number of colonies was determined. The results are in the following table [...] & Nkulengu rail; t:

For the test results follows, since & Nkulengu rail; the average number of colonies when heated in the presence of the [...] & Nkulengu rail; en [...] more reduced, than when heated without [...] & Nkulengu rail; em preparation.

Au & Nkulengu rail; Several units also comprise spores were tested on their response to the above, from B. stearothermophilus [...]. Each 120 °C [...]^{6 spores} were incubated with the 10 different lengths (above method 1) wherein. The 5 approaches have been tested temperature. One received the following results:

[...] from Y. pseudotuberculosis a substantially to speed up elimination of spores causes The.

[...] is made from vegetative cells of B. stearothermophilus A feature of more than on homologous to-coined/shaped effect on homologous spores in its vegetative cells. These [...] & Nkulengu rail is illustrated in the following table by the; th test results.

This attempt were used for each temperature^{6 10,} 10^5, 10^{4,3 10,} 10^2, 5 10^{1 5 10} 0 and ^{tests and spores of} B. stearothermophilus in control tests. [...] cells were used per temperature in amounts of^{7 from B. stearothermophilus [...],6 10,} 10^{5,4 10,} 10^{3,2 and1 5 10 10 5 tests and control test in cells}. It has been determined the total number of positive tests Q and P control tests. 3-hour exposure was a significant inhibition on the vegetative For already at a temperature of 85 °C to observe cells. 15-minute exposure by the the spores were [...] For at temperatures above 110 °C inhibited.

[...], in cultivation of bacillus subtilis, 168 and Bacillus licheniformis strain, strain G have been obtained, in combination with elevated temperature inhibitory effect on spores of B. stearothermophilus.

The above results show, since & Nkulengu rail; spores [...] heterologous vegetative cells can be inhibited also from.

Microorganisms, which do not form spores, are also sources of [...]. plague microbe Yersinia pestis For example, there are for from cells of, the inhibit are cultured at 28 °C, isolatable [...], the spores of B. stearothermophilus at 120 °C.

These results confirm, since & Nkulengu rail; [...][...]more nothing-pore-forming bacteria also may be a source of cells.

Escherichia coli, strain K -12 was cultured analogously to example 2. By washing the culture, centrifugation and heating the thus obtained was made to its activity against homologous [...] and a washing phase tested vegetative cells. Vegetative^{cells at 10} 2 52 °C [...] The expenditure in each case 30 minutes with the incubated at various dilutions. One received the following results:

Au & Nkulengu rail; [...][...] was compared with from E. Several units also comprise the (spore former) and Y. pseudotuberculosis from B. subtilis coli ( [...] ) with respect to their activity against vegetative cells from E. coli and spores of B. stearothermophilus. The results are in the following table [...] & Nkulengu rail; t:

The two groups of 5 - (5-FU) [...][...][...]pro medication were given with a or with a The liposome preparation, containing the same amount in combination with a 5-FU [...] & Nkulengu rail; en [...] -Preparation (1:1 mixture of [...] according to & Nkulengu rail; example 2 and 3). The tumor patients were given [...] in an amount of about 100 to 500 micrograms per application. 5-75 mg per application was in an amount of administered FU.

Both patients were subjected to-groups as well as an experimental pro medication After the control group a local hyperthermia-treatment. This hyperthermia equipmenttumor region has been heated by means of a the (Jasmin, [...] - [...] ) 1 hour and 40 minutes to a temperature of about 43.5 to 47 °C.

The treatment success was radiologically examined. The results are in the following table [...] & Nkulengu rail; t.

Trying 54 mice have been performed on (Hybrid F1 (CBAxC57b1/6), male, weight 22-24 g, age 3-6 months). Cells from^{Lewis lung carcinoma were transplanted per 10} 6 mice Den (strain LLC) into the right [...]. The tumors had reached a volume of 8 days 340 ± After 50 mm^3. The test animals were divided into 3 groups of a multiplicity of 18 (V1 and V2 a control group K and two test groups) mice. The test groups were anesthetized by administering an [...] mice The (70 mg of active ingredient per kg body mass). 5 to 7 min after the injection the animals were inserted into a [...], in which the lower body half via a thermostatic water bath could be heated (42.5 °C water temperature). The hyperthermal treatment lasted 30 min. While the first V1 has been subjected to only one hyperthermy treatment experimental group, the animals were given the V2 30 min before the hyperthermy treatment a [...]experimental group (i.v., per 0.4 g per mouse, 2-fold dilution of the starting solution, [...] from a mesophilic bacterium, according to prepared & Nkulengu rail; example 2).

The tumor volume was checked periodically to thermal bio tables After the treatment in & Nkulengu rail; strength intervals. The test animals were killed 21. day Am, the tumors were taken and [...] - lung solution fixed in. Au & Nkulengu rail; the number was determined on the lung surface metastases Several units also comprise the under a microscope in the test animals.

The test results are compiled in the following table.

For the experimental data is evident, since & Nkulengu rail; already the sole hyperthermal treatment (V1) a relevant [...] & Nkulengu rail; has on the primary tumor or the lung metastasis. Surprisingly, could be noted, however, since & Nkulengu rail; an injection of 30 min before the hyperthermy treatment leads to a significant tumor damage[...]. Also the average number of metastases in the lung is significantly reduced (V2).