DE102005011111A1 - Pharmaceutical composition comprising dendritic cells, useful for treating tumors, especially of the skin, where cells are loaded with animal toxins, formulated with a homeopathic component - Google Patents

Abstract

A pharmaceutical composition (A) for treating tumors comprising dendritic cells (DC) loaded with animal toxins, is new. A pharmaceutical composition (A) for treating tumors comprises dendritic cells (DC) loaded with: (i) toxins (T1), and/or associated synergists and/or antagonists, from the poison cocktail of a centipede of the genus Scolopendra, using as solvent in 15 ml of 0.9% saline solution, 7 ml of the homeopathic substance tarantula D4; and (ii) added to this mixture, up to 0.5 ml of a saturated solution of the total poison cocktail of a spider of the species Loxosceles laeta, L. gaucho, L. mallorca or L. menorca. Depending on the required ratio of component amounts, the total poison cocktail is ground in anhydrous formic acid, then1-2 ml of the total extract of poisons from bulldog ants (Myrmicinae) added, where this quantity refers to 10 ml total poison cocktail. ACTIVITY : Cytostatic. No biological data given. MECHANISM OF ACTION : The animal toxins have necrotic, cytotoxic and/or apoptotic properties.

Description

The The invention relates to dendritic substances loaded with toxic substances Cells, process for their preparation, and the use of these dendritic cells for the treatment of tumors, in particular for therapy of tumors of the mucous membrane and the skin.

Under Skin cancer is understood on the skin, more rarely on the mucous membrane, occurring tumors. The malicious Tumors are called skin carcinomas or melanomas. The primary tumors are manifested in the skin and on the skin surface as pigmented surfaces (Veronesi U., Cascinelli N., Santiani M. (1987); Cutaneous Melanoma. Status of Knowledge and Future Perspective. London, Orlando, San Diego, New York, Austin, Boston, Sidney, Tokyo, Toronto: Academic Press). To get them from birthmarks, with them often because of their appearance is a tissue sampling and to be confused in many cases Cultivation in tissue culture required. The most aggressive among the known malignancies is malignant melanoma, which is often called "black Cancer "is called. One problem with this condition concerns its extremely metastatic Propagation, which stops before any organ border.

The The following therapies are currently preferred.

operational distance

The common Procedure currently represents the surgical removal of skin cancer Because of the depth or the spread of skin cancer is one adequate dissection of the skin cancer is not possible. Therefore often becomes one Combination with other forms of therapy (Voigt H. (1996): 150 questions and answers about malignant melanoma. Zuckerschwerdt publishing house, Munich).

disadvantage

Inpatient stay and / or narcosis, wound healing and scarring

radiotherapy

nowadays Radiation therapy is decreasing in importance in skin cancer mainly only used for lymph node and skin cancer metastasis treatment. The irradiation takes place with ionizing radiation; usually One uses electron, gamma, neutron or X-rays (Zetkin / Schaldach: Lexicon of Medicine, 16th edition 1999, page 1922/1923 Ullstein Medical).

disadvantage the radiation is that, similar as with chemotherapy, a spatial limitation is not possible. Due to the radiation hardness Healthy cells are sustainably damaged, especially the DNA. There Cancer cells usually become faster than healthy cells divide the cancer cells under normal circumstances during radiotherapy killed first. However, there is a risk of Strahlenulkuses (Pschyrembel-Clinical dictionary 256th edition 1990, page 1602).

chemotherapy

In chemotherapy, metastatic melanomas, in particular, are hardly chemoresponsive. A monotherapy with cytostatics is usually not sufficient, so especially polychemotherapies are used. It is mainly the following substances that find an application:
Dacarazine (DTIC)
Cisplatin (CDDP)
Carboplatin (CBDCA)
Ifosfamide (IFO)
fotemustine
bendamustine
CCNU
BCNU
Methyl-CCNU
Cyclophosphamide (CPA)
Vindesin (DVA or VDS)
Vinblastine (VBL)
Vincristine (VCR)
Procarbazine (PBZ)
Mitomycin (MMC)

The application of these substances takes place in the following therapy protocols, which represent the currently most effective:
Bleomycin + Vincristine + CCNU + Dacarbicine (Bold Protocol)
BCNU + cisplatin + dacarbazine + tamoxifen (+ interleukin-2 / interferon-alfa)
Cisplatin + vinblastine + bleomycin (CVB protocol)
Cisplatin + Vindesin + Discarbazin (CVD protocol)
BCNU + hydroxyurca + dacarbazine (BHD protocol)
Dacarbazine + vincristine + BCNU (DVB protocol)
Cisplatin + ifosfamide
Fotemustin + interferon alpha
- Voigt H. (1996) 150 Questions and Answers on Malignant Melanoma Zuckerschwerdt Verlag, Munich -

Next show the positive effects against the genetically defective cells the mentioned chemotherapies unfortunately also have many side effects.

disadvantage The chemotherapies are that due to their chemical structure are difficult to use targeted; These cytostatics are extremely aggressive Cellular toxins are, in addition to tumor tissue also largely healthy Tissues, including Damage liver and kidney cells. The occurring side effects, such as Hair loss, dizziness, Vomiting, gastrointestinal bleeding, Circulatory problems, etc., are due to systemic spread The cytostatic drugs are difficult to weigh (German Cancer Research Center DKFZ Heidelberg - Focus 19/1995).

These numerous. dangerous and unwanted Explain side effects especially from the regeneration inhibition rapidly proliferating Tissues and especially affect the hematopoietic system, the epithelia mucous membranes and the gonads, as well as the skin and skin appendages. Among the life-threatening Complications are top of the list, followed by infections Bleeding (Pschyrembel - Clinical dictionary 256th edition 1990, page 1866).

cytokine treatment

Mainly tried one here only for Indications of Kaposi's sarcoma and hair cell leukemia approved interferon therapies apply. Interferon belongs active cytokines active against degenerated cells. The mode of action is based on the detection of surface proteins of cancer cells and their destruction. The disadvantages of interferon therapy include fever, anorexia-nausea-emeses syndrome, Fatigue, headache, headache, joint pain, dizziness, Liver enzyme increase, Pruritus, IIN antibody formation, Circulatory and blood pressure problems (De Vita jr. VT., Helman S., Rosenberg SA. (1991): Biologic Therapy of Cancer Philadelphia: J.B. Lippincott Co.).

Antibody therapy

Next Interferon therapy is used in immunotherapies in the Treatment mainly on the use of monoclonal antibodies. To become myeloma cells grown by a method of Köhler and Millstein fused with rodent lymphocytes, which interact with human antigens Melanoma cell material or Membanfraktionen from melanoma cells immunized were. This produces almost unlimited amount of monoclonal antibodies Melamomzellen. The antibodies are then injected and then find naturally via immunomodulators the skin cancer cells and destroy these. Side effects are not known since they are the body's own Substances, so that no immune shock reactions occur. The disadvantage of this form of therapy, however, is the low sensitivity to skin cancer cells (Voigt II. (1996): 150 questions and answers on malignant melanoma. Zuckerschwerdt Verlag, Munich / Ibelgaufts N. (1992): Lexicon Zytikinc. medicon Munich).

Therapy with dendritic cells

Since about 1995, the possibility of using endogenous dendritic cells in the treatment of skin cancer has been intensively researched. For this purpose, spinal cord and / or blood stem cells of the Pa dendritic cells were cultured in vitro, which, when they reached a minimum titer, are then re-vaccinated to the patient. The dendritic cells actively seek and find tumor cells and attack them with cell-destroying substances. Here, the low efficiency is the main drawback, so that one tries to load the dendritic cell with the body's own human cytokines and / or peptides (eg anaphylatoxins C3a and others) (Kirchner H., Kruse A., Neustock P., Rink L. (1994): Cytikines and Interferons, Spektrum Akademischer Verlag, Heidelberg). Unfortunately, the specificity of the substances found is often insufficient to completely kill the tumor cells.

task It is therefore an object of this invention to provide improved means for the Therapy of tumor diseases by means of dendritic cells are vulnerable. The effect of these funds should continue to be improved special solvents elevated become.

These Task is achieved by achieves the loading of dendritic cells with toxic substances, whereby the toxic substances from the venom of scolopenders of the Genera Scolopendra and Hemiscolopendra, snakes of the genera Bitis and Naja, spiders of the genera Loxosceles, Sicarius and Pholcus, as well as scorpions of the genus Parabuthus and a combination of one or more of these toxins are selected.

• 1. Als Basisbestandteil werden in 15 ml 0,9 prozentiger NaCl – Lösung 7 ml der homöopathischen Substanz Tarantula D4 eingearbeitet.

In this case, the solvent according to the invention is composed of several constituents:

• 1. As a basic component, 7 ml of the homeopathic substance Tarantula D4 are incorporated into 15 ml of 0.9% NaCl solution.

Tarantula D4 is mixed on homeopathic Shake well to the center of the earth with 15 ml of the 0.9% NaCl solution.

As is known, in the homeopathy the patient does not receive the drug in question in his mother tincture, but in a dilution stage. The founder homeopathy, Samuel Christian Hahnemann made the seemingly paradoxical observation that the effect of a drug is inversely proportional to the concentration behaves. The stronger the stock solution dilute the more effective it becomes.

• 2. Zu der unter 1. erhaltenen Lösung gibt man bis zu 0,5 ml einer gesättigten Lösung des gesamten Giftcocktails von Spinnen der Arten Loxosceles laeta, oder Loxosceles gaucho, oder Loxosceles Mallorca, oder Loxosceles Menorca.

The D-dilution steps are prepared by adding 1/10 of the stock solution to 9/10 alcohol and then shaking. This gives the first dilution D1. Take another 1/10 of this dilution and shake with 9 parts of alcohol to obtain a D2. So this procedure is continued until finally high powers such as D 200 arise. Nevertheless, these are highly effective medicines. However, in order to search for an explanatory model, one has to say goodbye to physics, which works with simple linear substance-related theses.

• 2. To the solution obtained under 1. add up to 0.5 ml of a saturated solution of the total poison cocktail of spiders of the species Loxosceles laeta, or Loxosceles gaucho, or Loxosceles Mallorca, or Loxosceles Menorca.

From of the species Loxosceles are in the warmer up to the tropical areas of North America, Central America, South America and the Caribbean about 50 species represented.

In Africa to the Mediterranean and Southern Europe are about 20 species to find from Loxosceles.

The mostly in brown tones colored Animals reach a length of the body (without legs) from 8 to 15 millimeters. These spiders are mostly Active at night and very shy. Your network is irregular. As a shelter, they seek out dark places under stones or also under foliage. Loxosceles laeta is mostly found in cellars, garages, Sheds, stables or even in the apartments themselves. You have to assume that the room temperatures in the buildings made it possible that this species spread so far, even in colder regions could.

Loxosceles - species bite only in strong physical Contact.

Especially in the living area, the bites are registered, the symptoms similar to a stinging bug have a stitch. There is the concept of "necrotic arachndism". This describes skin necrosis after bites of the Loxosceles species.

• 3. Aufgrund unterschiedlicher Mengenverhältnisse der verwendeten Gesamtgift – Cocktails ist es teilweise erforderlich, den Gesamtgift – Cocktail in wasserfreier Ameisensäure anzureiben, der wiederum 1 bis 2 ml des Gesamtextrakts von Bulldog – ants zugemengt wird. Diese Menge bezieht sich auf eine Menge des Gesamtgift – Cocktails Von 10 ml.

Necroses are also known after bites from other spiders.

• 3. Due to differences in the proportions of the total poison cocktails used, it is sometimes necessary to coat the whole poison cocktail in anhydrous formic acid, which in turn is added to 1 to 2 ml of the total extract of Bulldog ants. This amount refers to an amount of the total poison cocktail of 10 ml.

The Bulldog - Ants are ants that belong to the family of Myrmicinae.

she live in Australia. There are many types of them and they are the biggest ants on earth. You can a length of up to 40 millimeters. They are diurnal and come in different sizes and Colors in front.

The Bulldog - ants have a long head, big Eyes and strong Jaw. They often attack in larger numbers and her bite is poisonous. Thirty of these ants can do one Kill people.

A different kind of bulldog - ant is small, black and moves forward jumping. These Art creates jumps in a height to 20 centimeters.

In the solvent thus obtained will then be for the particular application of the specific pharmaceutical active substance incorporated.

To The previous findings are the solvents described not toxic.

The solvent alone have a tumor destroying Effect of cell experiment in brain tumors, gastrointestinal tumors and individual breast cancer patients - cell lines were tested positive.

so loaded dendritic cells are used to treat tumors, in particular for the treatment of skin cancer and mucosal tumors, used. The dendritic cells can be used as sole therapy, but also as an adjunctive therapy without the risk of the disadvantages of current state of the art.

following the invention will be closer described. However, the invention is not limited to the following Description and the embodiments limited. The person skilled in the art can claim the invention on the basis of the attached patent claims consideration modify the description without changing the scope of protection of the enclosed claims to leave.

The Loading of the dendritic cells takes place with one and / or more toxic substances from the venom of scolopenders of the genera Scolopendra and / or Hemiscolopendra, snakes of the genera Bitis and well, as well as scorpions of the genus Parabuthus and / or spiders of the genus Loxosceles, Sicarius and Pholcus, in a pharmaceutical and therapeutically effective amount. But it is also possible a combination of at least two of these toxic substances from the poisons.

The Gift of the above animals contains a whole cocktail of Compounds, which include not only peptide toxins but also toxins an amido group, which, however, have no protein structure and for example, have a molecular weight of 30-55 kDa. Farther belong to the poison cocktail antagonistic substances that a spatial and temporally controlled spread of the animal's output Ensure poisons.

The toxins are preferably peptide toxins. The toxins generally have necrotic, cytotoxic and / or apoptotic properties. The toxins can be isolated from the poison cocktail of the animals by methods known per se. However, since this is cumbersome, it has been found according to the invention that, after fractionating the poison cocktails, it is also possible to use individual fractions containing the toxins for loading the dendritic cells. In these fractions, the toxins are present together with other substances. However, since the fractions are selected so that they contain, in particular, the cytotoxic, apoptotic and / or necrotic substances, purification of the toxins up to complete purity is not absolutely necessary. However, if this has been done and, for example, the structure of the toxins is known, they could be present not only in isolated, pure form but also in recombinant form be used when it comes to the peptide toxins. The peptide toxins generally have a molecular weight in the range of 50-350 kDa, preferably a molecular weight of about 100 kDa. The toxins can also be produced synthetically, and it is of course also possible to modify the toxins present in the abovementioned species and used according to the invention for loading the dendritic cells, ie derivatives obtained by chemical modifications of the toxins. In the case of the peptide toxins, derivatives include, inter alia, those toxins in which one or more amino acids have been added, deleted and / or replaced by other amino acids, whereby of course the toxic properties are retained. It should be noted that a toxin is not only cytotoxic, but may also combine necrotic and / or apoptotic properties.

The with the toxic substances loaded dendritic cells can targeted therapy of tumors can be used. Consequently, show the toxins cell-destroying Properties on. Because the dendritic cells actively activate tumor cells search and find and attack them with cell destructive substances, the toxic substances selected so that they are cell-destroying, i.e. cytotoxic, necrotic and / or apoptotic properties exhibit. Just such substances are in the poison cocktails the animal species mentioned in claim 1. They are therefore suitable in an excellent way for the treatment of tumors of the skin and the Mucous membranes. But it is also possible the thus loaded dendritic cells with further, for the tumor therapy to load usable substances, for example with cytokines, Cytokine receptors, anaphylatoxins, interferons, antibodies, glycosides etc.

The Loading takes place, for example, by direct injection of the toxic Substances into the cell, by incubation of the dendritic cells with the toxic substances or by lipofection. Originally acted this is a technique for the transformation of cells with foreign DNA or RNA. increase effectiveness is achieved by liposomes antibodies, proteins, glycolipids et al linked Targeting the interactions of liposomes and cell surface to be influenced. Thereby can the peptide toxins or cytokines are recognized as "cell-like" and in the "normal" metabolic functions into the dendritic cells (Herder (1995): Encyclopedia of Biochemistry and Molecular Biology, Volume 2, Spectrum Academic Verlag Heidelberg).

One Another method of loading is laser microinjection. there be dendritic cells, which in one the toxic substances containing medium, for treated for a short time with a highly focused laser beam, so a hole is created in the cell wall through which the surrounding medium can penetrate into the cell. The underlying this method Technique of micromanipulation of cells or cell aggregates eg in Sagittarius K. et al .; Nature Biotchnology, 16,8: 737-742 (1998), Sagittarius K. et al .; Biol. 55: 735-746 (1998), Bohm M. et al .; Americ. J. Pathology; 151, 1: 63-67, Ponten F. et al .; mutation Research Genomics, 382: 45-55 (1997), Bernsen M. et al .; Lab. Invest. 78: 1267-1273 or Fink L. et al .; Nat. medicine. 4,11: 1329-1333 (1998).

Prefers it is when the peptide toxin that loads the dendritic cell is, and / or the optionally further contained substances the poison of the Skolopender dogfish species Scolopendra gigantea ssp., Hemiscolopendra spp., The snake species Bitis arietans (Puffotter), Bitis gabonica (Gaboon otter). bitis nasicornis (Rhino viper) and the smaller species B. atropos, B. caudalis, B. peringueyi and the Speichobraarten Naja melanoleuca, Naja pallida and Naja sputatrix, the parabuthus scorpion species Parabuthus transvallicus, Parabuthus granulosus, Parabuthus villosus, the Loxosceles spider species Loxosceles laeta, Loxosceles spiniceps, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens. Loxosceles reclusa, Loxosceles deserta, the six-eyed crab spider species Sicarius hahni, Sicarius albospinosus, Sicarius oweni, Sicarius testaceus, Sicarius argentinensis and / or the dormouse species Pholcus phalangioides, Pholcus opilionides, Pholcus spp. (RSA) and Pholcus spp. (Cuba) come, whereby it particularly preferred is when the peptide toxin and / or optionally further Substances from the poison of Skolpenders, Bitis arietans and / or Loxosceles spiniceps come. This has the advantage that at the Substance is the natural skin and / or tissue-degrading substance Effect can be exploited. It is preferred that the animal poison raw mixture from male and / or female Skolpendern of the genera Scolopendra and Hemiscolopendra from about 15 cm body length, male and / or female snakes of the genera Bitis and Naja, preferred from a body length of 50 cm, female subadult and / or adult scorpions of the genus Parabuthus, and / or female subadult or adult spiders of the genus Loxosceles and / or Pholcus and / or Sicarius becomes. This is beneficial because female arachnids of the genera Parabuthus, Loxosceles, Pholcus and Sicarius produce more venom as a male Representatives. In scolopenders and snakes, both sexes produce comparable poison quantities and poison qualities. It is further preferred that the animal poison raw mixture by manual Milking is obtained. This has the advantage of a particularly gentle Obtaining the animal poison raw mixture.

at the method according to the invention is moreover preferred that the raw animal poison mixture homogenized prior to fractionation is preferred, and it is further preferred that the fractions before the Further processing deep-frozen and more preferably lyophilized. The pharmaceutical according to the invention Loads of dendritic cells are suitable for use in the medicine. The loaded with the pharmaceutically active substances dendritic cells can preferred according to the invention used to treat skin cancers. Prefers is still the use of a peptide toxin from the venom of Centipede-Hunderfüsserarten Scolopendra gigantea ssp., Hemiscolopendra spp., The snake species Bitis arietans (Puffotter), Bitis gabonica (Gaboon otter). bitis nasicornis (Rhino viper) and the smaller bitis B.atropos, B.caudalis, B.peringueyi and the Speichobraarten Naja melanoleuca, Naja pallida and Naja sputatrix, the parabuthus scorpion species Parabuthus transvallicus, Parabuthus granulosus, Parabuthus villosus, the Loxosceles spider species Loxosceles laeta, Loxosceles spiniceps, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens. Loxosceles reclusa, Loxosceles deserta, the six-eyed crab spider species Sicarius hahni, Sicarius albospinosus, Sicarius oweni, Sicarius testaceus, Sicarius argentinensis and / or the dormouse species Pholcus phalangioides, Pholcus opilionides, Pholcus spp. (RSA) and Pholcus spp. (Cuba) in a pharmaceutical effective loading of dendritic cells for the treatment of skin cancers, wherein peptide toxins from the venom of scolopenders, Bitis arietans and / or Loxosceles spiniceps are particularly preferred.

Besides that is the use of a peptide toxin from the venom of Scolopender dogfish species Scolopendra gigantea ssp., Hemiscolopendra spp., the snake species Bitis arietans (Puffotter), Bitis gabonica (Gaboon otter). bitis nasicornis (rhino viper) and the smaller bitis B.atropos, B.caudalis, B.peringueyi and the Speichobraarten Naja melanoleuca, Naja pallida and Naja sputatrix, the Parabuthus scorpion species Parabuthus transvallicus, Parabuthus granulosus, Parabuthus villosus and / or the Loxosceles spider species Loxosceles laeta, Loxosceles spiniceps, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens. Loxosceles reclusa, Loxosceles deserta and the Pholcuss spider species Pholcus phalangioides, Pholcus opilionides, Pholcus spp. (RSA), Sicarius hahni, Sicarius albospinosus, Sicarius testaceus, Sicarius oweni and Sicarius spp. (Southern Maerika) provided in medicine according to the invention.

Prefers For example, the peptide toxin is obtained by a fractionation method and it is further preferred that the pharmaceutically active Substance is obtained from the poison cocktail of one of the species mentioned. As a result, the pharmaceutical effectiveness of the loading in their Effect advantageously on the type of tumor to be treated and / or Size matched become.

The Peptide toxin can be prepared by fractionation methods known per se for the separation of proteins from the raw animal poison mixture (Gesamtgiftcockail) to be obtained. It is preferred that the peptide toxin be purified by gel chromatography, HPLC, affinity chromatography and / or ion exchange chromatography. But it is also possible, the peptide toxin and / or the other substances used the poison of Skolopender dogfish species Scolopendra gigantea ssp., Hemiscolopendra spp., The snake species Bitis arietans (Puffotter), Bitis gabonica (Gabon-otter). bitis nasicornis (Rhino viper) and the smaller bitis B.atropos, B.caudalis, B.peringueyi and the Speichobraarten Naja melanoleuca, Naja pallida and Naja sputatrix, the parabuthus scorpion species Parabuthus transvallicus, Parabuthus granulosus, Parabuthus villosus, the Loxosceles spider species Loxosceles laeta, Loxosceles spiniceps, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens. Loxosceles reclusa, Loxosceles deserta, the six-eyed crab spider species Sicarius hahni, Sicarius albospinosus, Sicarius oweni, Sicarius testaceus, Sicarius argentinensis and / or the dormouse species Pholcus phalangioides, Pholcus opilionides, Pholcus spp. (RSA) and Pholcus spp. (Cuba) chemically synthesized or by recombinant genetic engineering methods Mold. As usual with chemicals the present invention also provides derivatives and salts of the invention provided Substances. For example, the peptide toxin may be one or more Include additions, substitutions and / or deletions of amino acids, it must be ensured that the pharmaceutical-medical Effect is maintained.

Prefers is also that the peptide toxin in such an amount to the pharmaceutically effective dose is present for loading, that by the dendritic cell only one controlled delivery of the peptide toxin takes place.

Farther preferred is a pharmaceutically active loading in which the Amount of peptide toxin that the dendritic cell can deliver, in their cell-destroying Effect spatially and temporally subject to a controlled spread. The pharmaceutical Composition preferably has an amount of peptide toxin which dependent on is selected from the skin cancer and / or other tumor to be treated.

Raising the dendritic cells

Prefers are the human spinal cord stem cells deep-frozen in Eppendorf sample cups (E-cups) with a capacity of about 1 ml. This is preferably a saturated stem cell solution (saline or Plasma, maximum 50 microliters). For further processing are preferred 750 μl medium (37 degrees Celsius), consisting of 500 ml DMEM-Hams's F-12, 50 ml RPMI 1260 penicillin / streptomycin, 5 ml glutamine and 50 ml FBS added.

The so diluted Suspension is subsequently 10-15 Seconds at the lowest level on the Vortex without foaming shaken to homogenous mixing.

Immediately thereafter, the suspension is placed in a 75 cm ² cell culture flask and filled with 20 ml of the same medium (37 degrees Celsius).

Then There is a slight two to three times manual panning for optimal distribution of the spinal cord stem cells in the suspension in the cell culture bottle.

These Cell culture flask is set to 37 degrees Celsius Incubator introduced.

Prefers It is the set cell culture for five days at the same Temperature (37 degrees Celsius) in the incubator to leave.

In order to becomes a firming of the spinal cord stem cells on the bottom of the bottle. The first visual check under the inverted microscope, whether the spinal cord stem cells grown on the bottom of the bottle is preferably five days after placing the cell culture flask in the incubator.

It is preferred to carry out the first medium change under sterile conditions as follows, five days after preparation of the cell culture:
The medium bottle is held so that as much of the medium contained in it is poured out without flushing out the already grown cells. Possibly. remaining residual medium is removed by gently tapping the bottle neck on a pad.

The so collected medium is discarded.

Then be Carefully introduce 20 ml of new medium into the cell culture flask, without detaching the grown cells from the cell culture bottoms. Of the so described medium change is in each case after three or four days until several layers of cells have grown over each other and the first cells are protruding Loosen the medium.

The Growth of the cell culture is controlled under the Ivert microscope before each medium change.

When the multi-cell cell bottle is grown on the cell culture bottoms, the entire supernatant of about 20 ml is subcultured in equal parts in 4 small 25 cm ² bottles and then filled with 37 degrees Celsius medium.

These Subcultures become five Days in the incubator at 37 degrees Celsius left without further activities.

From the fifth Day takes place every three or four days of the previously described Change medium with 5 ml of medium under running microscope control.

If the cell culture bottle bottom completely covered with cell layers is the addition of the growth factor mixture in the amount, that a concentration of 900 U / ml GM-CSF and IL-4 as well as 700 U / ml sIL-4R (e.g., from Biochrom). Is preferred while a temperature of 37 degrees Celsius. The addition of the growth factor mixture by means of a sterile 2 ml pipette.

The Cell culture flask is easily panned manually without the cells to be detached from the ground. For at least The cell culture flask is kept in the incubator at 37 degrees Celsius for 24 hours stored.

At the earliest after 24 hours, the first visual inspection under the inverted microscope takes place, whether it is already Dendritic cells have been differentiated, which are then transferred each day under the most sterile conditions by means of a glass pasteur pipette with the least possible medium in a new bottle (25 cm ² ) with already heated to 37 degrees Celsius medium (2 ml) until in the original bottle no longer form new dendritic cells.

Of the Medium change for the dendritic cells collected in the new cell culture flask is preferably made in the way that the old medium until to 1 ml with a glass Pasteur pipette under the most sterile conditions is aspirated without dendritic cells with suction and then is filled with 4 ml of fresh, 37 degrees Celsius medium.

This Medium change is then every three or four days made in the manner described.

Due to the dendritic cells thus obtained having a viability of at least three weeks, a sufficient number are available for therapeutic purposes. Action:
The dendritic cells recognize genetically defective endogenous cells, dock on them and release the added active substance into the diseased cell. The loading causes the targeted destruction of the degenerate cell on the cytotoxic effect. In addition, dendritic cells are able to transmit information about tumor events (through detection of cytokines), but also information about tumor-destroying substances to T cells. The "informed" T cells are called killer cells.

advantage of it is that the dendritic cells are able to genetically Detecting defective cells, the load enclosed accurately to bring the site of action and prevent the formation of daughter ulcers.

description

Next about 1500 worldwide in temperate zones occurring species of scorpion and the approximately 35000 Spider species are known only of about 70 species of scolopops. With the exception of about 300 spider types belong the aforementioned animals all to the actively poisonous animals, theirs Use poisons for food purchase. Because these animals are only a very small mouth opening have developed the external pre-digestion, the above a sophisticated enzyme and poison system is made possible. Either will inject a poison cocktail directly into the prey or the prey is killed mechanically, torn and / or durchgekaut and mixed with the poison cocktail. The liquefied Food with the smallest solids can then be absorbed become.

• – Verdauungsenzyme
• – Biogene Amine
• – Organische Säuren
• – Peptide
• – Peptidtoxine

About 50 species of spiders, as many scorpion species and about 20 species of scollopids can also be dangerous to humans through their poisons. Above all, the poisons of the medically important species are hardly or not at all researched. The main components of the Arthropodengifte are:

• - digestive enzymes
• - Biogenic amines
• - Organic acids
• - peptides
• - peptide toxins

• – Herzgifte
• – Nervengifte
• – Blutgifte
• – Zellgift
• – Gewebe zerstörende Gifte

Among the peptide toxins are the following toxin groups:

• - heart poisons
• - Nerve toxins
• - Blood toxins
• - cell poison
• - tissue damaging poisons

Originally finds through the total poison cocktail of all the poisonous animals by the synergistic and / or antagonistic interaction of different Substances generally also have a pre-digestion and thus a targeted Source cells change animal cells instead.

In the scolopender, snake, scorpion and spider species used in this invention, substances which are cytotoxic, necrotic and / or apoptotic (digestive action of the poisons) are found in the total poison cocktail. On the other hand, substances which act as cardiac poisons, neurotoxins or blood poisons should rather be avoided according to the invention. By HPLC-MS-MS were the peptide toxins from the total poison cocktail. These in turn were assigned specific modes of action via cell experiments. Surprisingly, it has now been found that constituents of the animal poisons of Scolopender centipede species Scolopendra gigantea spp., Hemiscolopendra spp., The snake species Bitis arietans (Puffotter), Bitis gabonica (Gabunotter). bitis nasicornis (rhino viper) and the smaller bitis B.atropos, B.caudalis, B.peringueyi and the Speichobraarten Naja melanoleuca, Naja pallida and Naja sputatrix, the Parabuthus scorpion species Parabuthus transvallicus, Parabuthus granulosus, Parabuthus villosus, the Loxosceles spider species Loxosceles laeta, Loxosceles spiniceps, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens. Loxosceles reclusa, Loxosceles deserta, the six-eyed crab spider species Sicarius hahni, Sicarius albospinosus, Sicarius oweni, Sicarius testaceus, Sicarius argentinensis and / or the dormouse species Pholcus phalangioides, Pholcus opilionides, Pholcus spp. (RSA) and Pholcus spp. (Cuba) can be used for the treatment of skin cancers, with peptide toxins from the venom of scolopenders, Bitis arietans and / or Loxosceles spiniceps are particularly preferred.

Of the Total poison cocktail is due to the large number of individual substances contained and its resulting complex effect in the body currently pharmaceutically not yet usable. As a defense poison from the animals Used, suffered the bite the following symptoms;

Skolopender of the genera Scolopendra and Hemiscolopendra

On Reason of his great Mouthparts will bite the animals up to 30 inches long, always felt. General Observations about the poisonous effects of Giant Centipedes (Skolopender) are missing. The poison is definitely for small mammals highly effective. That's enough for example, the contents of a venom gland around 25 mice weighing about 20 gr. Kill within 7 hours. The poison has a strong effect the nervous system, leading to the following symptoms: acceleration of the breath, Sweating, Incoordination, Vomiting, respiratory paralysis, cramps until death. Recent work on There is no chemistry, biochemistry and toxicology of poisons (Habermehl G. (1987): Poison Animals and their Weapons Springer-Verlag, Berlin, 4. and subsequent editions).

snakes of the genera Bitis and Naja

Of the Bite itself is from the bitten by the force with which the Serpent happens to be perceived. As a special feature is the ability to mention the Speikobras, the above her teeth by means of compressed air the poison purposefully up to 5 meters on the Spraying victims can. With the bite continues the gift of immediate pain. Soon after, the poison begins with its tissue-degrading effect. First, the local necrotic begins Effect of lysing the dermal layers. Then spreads themselves with the help of the permeability-increasing Enzyme the poison in the large area the bite site surrounding tissues. It can except the skeleton all tissue fixtures over Necrosis destroyed become.

He follows the bite in a limb Amputation must be weighed according to the situation. Next to the tissue-destroying poisons Depending on the species, you will still find neuro- or cardiotoxins that are in the first place Line for the pain and circulatory instability are responsible. at the deadly running bite accidents Blood poisoning and kidney failure come first.

scorpions of the genus Parabuthus

Nice the sting caused due to the neurotoxins contained in the poison cocktail severe pain at the sting site, which resembles the pain of a burn. The Pain is replaced after some time by a tingling which ultimately turns into a numbness. The general symptoms can be after 5 minutes, but sometimes only 24 hours after the sting occur. The nerd is excited, children occasionally Cramps; Reflexes go back to a minimum or disappear altogether. The Awareness is maintained, often with severe anxiety. It follows tears and dilatation of the pupils while restricting the Eyesight. The pulse is accelerated and irregular. Blood pressure can be both elevated be humbled as well. The body temperature varies greatly, with hypothermia worsening State of the patient indicates. The breath is irregular. Vomit is a serious sign that also nerve centers are attacked. Mostly death occurs respiratory paralysis one, usually within 20 hours, less frequently within 30 hours (Habermehl G. (1987): Poison Animals and their Weapons, Springer-Verlag, Berlin, 4th and subsequent editions).

Spiders Genus Loxosceles

Of the Bite itself is not felt in most cases by the bitten. The Bite swells very strong after 2 to 5 hours, the Swelling is associated with severe pain. The bite discolored dark red and bubbles form. Subsequently, the tissue turns black and skin cells die off. This leaves a hole in the skin, which can be up to 5 cm in diameter. The cure can up to 2 years, with partial skin grafting become necessary can. In severe cases it comes to blood in the urine and ultimately to kidney failure Death. There are also cases with hemolytic anemia and hematuria which with sensory disorders accompanied. The body temperature can rise to 41 degrees Celsius, which in severe cases, too can set a coma.

Spiders Genus Pholcus

Also here the bite is not felt in most cases. To about 2 hours redden the bite site becomes noticeable and wet. Over the next few hours dissolves the skin layer around the bite site up to a size of 3 cm. It forms an open wound, which without treatment up to 3 months not healed. Without treatment, wound healing takes place with severe scarring. A treatment form consists e.g. in a skin graft. Accompanying symptoms are expressed in a circulatory instability. Deaths are not known.

Surprisingly can however, individual substances, from the toxin toxins contained in the animal venom, which of Scolopender centipede species Scolopendra gigantea ssp., Hemiscolopendra spp., the snake species Bitis arietans (Puffotter), Bitis gabonica (Gabunotter). bitis nasicornis (rhino viper) and the smaller bitis B.atropos, B.caudalis, B.peringueyi and Species Naja melanoleuca, Naja pallida and Naja sputatrix, the Parabuthus scorpion species Parabuthus transvallicus, Parabuthus granulosus, Parabuthus villosus, the Loxosceles spider species Loxosceles laeta, Loxosceles spiniceps, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens. Loxosceles reclusa, Loxosceles deserta, the Six-eyed crab spider species Sicarius hahni, Sicarius albospinosus, Sicarius oweni, Sicarius testaceus, Sicarius argentinensis and / or the dormouse species Pholcus phalangioides, Pholcus opilionides, Pholcus spp. (RSA) and Pholcus spp. (Cuba), for treatment of skin cancers as well as parallel or supportive too Skin cancer surgeries are used to genetically degenerate cells to destroy. For example, according to the invention, the destruction of melanoma cells and / or of tumor tissue not detected during surgery beyond the tracing, Disarm and the targeted elimination of genetically defective Cells take place. The therapy can be genetically defective body cells (Tumor cells) destroyed because of the surface protein structure and the inventively used, with peptide toxin-loaded dendritic cells, these in their surface structure changed Detect and destroy cells can. About that In addition, tumor cells give specific messengers, so-called tumor markers to which the peptide toxin-loaded dendritic cells and then destroy the tumor cells (Zitvogel L. et al (1999): Novel Immunologic and Therapeutic Attributes of Dendritic Cells (DC): Modulation of Tand NK cell-mediated anti-tumor immune responses, The European Journal of Cancer, Vol. 35 Supplement 5-S25 // Gong J., Avigan D. et al (1999): Activation of Antitumor Cytotoxic T Lymphocytes by Fusions of Human Dendritic Cells and Breast Carcinoma Cells. The European Journal of Cancer, Vol. 35 Supplement 5-S30).

The functionality is based on the ability of dendritic cells to detect, detect and destroy genetically defective cells and the tissue-destroying effect of peptide toxins as follows:
The peptide toxins with a molecular weight of about 50-350 kDa, in the case of scorpions, spiders and some scolopenders typically about 100 kDa, have a tissue-destroying effect, including a skin cell type destructive effect. Due to their high molecular weight and their spatial structure, they have little tendency to spread in tissues without the aid of so-called permeation enzymes. Dendritic cells are preferably produced and isolated from spinal cord stem cells for their equality by means of a growth factor mixture (GM-CSF / IL-4, preferably in combination with the interleukin receptor sIL-4R).

The Thus obtained dendritic cells are preferred with the cutaneous necrotic effective column chromatography obtained individual components from the poisons of Skolopender Hundertfüsserarten Scolopendra gigantea ssp., Hemiscolopendra spp., The snake species Bitis arietans (Puffotter), Bitis gabonica (Gaboon otter). bitis nasicornis (Rhino viper) and the smaller bitis B.atropos, B.caudalis, B.peringueyi and the Speichobraarten Naja melanoleuca, Naja pallida and Naja sputatrix, the parabuthus scorpion species Parabuthus transvallicus, Parabuthus granulosus, Parabuthus villosus, the Loxosceles spider species Loxosceles laeta, Loxosceles spiniceps, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens.

Loxosceles reclusa, Loxosceles deserta, the six-eyed crab spider species Sicarius hahni, Sicarius albospinosus, Sicarius oweni, Sicarius testaceus, Sicarius argentinensis and / or the dormouse species Pholcus phalangioides, Pholcus opilionides, Pholcus spp. (RSA) and Pholcus spp. (Cuba) on lipofection loaded. The quantitative loading of dendritic cells can be increased by an about 1.66% addition of phenyl-Gal-Nac.

possibly can other substances contained in the raw animal poison mixture the effects the peptide toxins that load the dendritic cells, support.

The Mode of action of the dendritic cells loaded with peptide toxins by testing them in appropriate healthy and tumorous cell lines respectively. Originate according to the invention the toxic substances, or the peptide toxin, with which the dendritic cells are loaded from the venom of scolopenders the family Scolopendidrae, snakes of the family Viperidae, snakes the family Elapidae, spiders of the family Sicarüdae (according to the invention belong to the Family of Sicarüdae besides Sicanus also the genera Loxosceles and Scytodes), spiders the family Pholcidae, scorpions of the family Buthidae.

Prefers are the genera Scolopendra and Hemiscolopendra, Bitis and Naja, as well as Loxosceles, Pholcus and Parabuthus. In the Bereidh of the genera Scolopendra and Hemiscolopendra may be the Scolopendra and Hemiscolopendra Giant Caged Centipede species Scolopendra morsitans, Scolopendra gigantea and Hemiscolopendra spp., in the genus Bitis the Bitis snake species Bitis arietans; Bitis gabonica and Bitis nasicornis, in the genus Well, the Naja snake species Well melanolcuca, Naja pallida and Naja sputatrix, in the area of Genus Loxosceles the Loxosceles spider species Loxosceles laeta, Loxosceles spiniceps, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens, Loxosceles reclusa, Loxosceles deserta, in the area of Genus Sicarius the Sicarus spider species Sicarius hahni, Sicarius albospinosus, Sicarius oweni, Sicarius testaceus, Sicarius argentinensis, in the genus Pholcus the pholcus spider species Pholcus phalangioides, Pholcus spp. (RSA), Pholcus spp (Cuba) and Pholcus opilionides, in the genus Parabuthus the Parabuthus scorpio species Parabuthus transvallicus, Parabuthus granulosus and Parabutus villosus particularly preferred. Among the spiders of the genus Loxosceles are according to the invention also the other types can be used.

Under the scolopenders of the genus Scolopendra and Hemiscolopendra according to the invention all types with a body length from 10 cm can be used. Among the snakes of the genus Bitis are all Zwergpuffotternarten used according to the invention. Under the snakes The genus Naja according to the invention is also the Asian Speikobra Well atra applicable.

Under The spiders of the family Pholcidae are according to the invention all major species used.

Under the scorpions of the genus Parbuthus are also all according to the invention Can be used in Central African species. In addition, according to the invention are the scorpions of the genera Opistophtalmus, Androctonus and Nebeo.

The Preparation of the pharmaceutical according to the invention Effective loading of dendritic cells can be done in such a way that dendritic Cells a peptide toxin under the microscope directly over a Micropipillary is injected, and then these cells in the culture flask be returned. In this coinage can the already loaded with this peptide toxin dendritic cell their information about the uptake of a cytotoxin to the other dendritic cells pass on. As a result, after about 72 hours a solution of this used Peptidtoxins be added to the medium. After another For 48 hours, the dendritic cells have a specific one, from the peptide toxin and the dendritic cell dependent Amount of peptide toxin added.

The Production can also be carried out in such a way that a culture of dendritic Cells in which skin cancer cells are located, directly in the culture flask is inoculated with a peptide toxin destroying the skin cancer cells becomes. Because the poison will destroy intact cancer cells in tumor cell depletion certain brands free, which the dendritic Cells indicate that the tumor cell destruction with the help of peptide toxin can be done more effectively, with the adaptation of dendritic cells with the active substances over the addition of phenyl-Gal-Nac can be increased quantitatively.

As a result, a further, concentrated addition of the peptide toxin can take place after about 24 hours, which is then also absorbed by the dendritic cells. The aim of the production process is to achieve the highest possible quantitative loading of the dendritic cells with this peptide toxin. A determination of the concentration of the cell is currently technically impossible or very difficult. However, in the last incubation step, the medium concentrations of substance to be loaded usually proceed in the range from about 0.5 μg / ml to 250 μg / ml, depending on the toxin used.

Prefers the production of a mixture of raw animal poison can be done in such a way that this by methods known per se from the arachnids and scolopenders and a fractionation of the raw animal poison mixture by also known per se fractionation process for separation of proteins is made to the peptide toxins and / or the other cell destructive acting substances in as possible from each other get separated form in separate fractions. For the production a pharmaceutically effective loading of dendritic cells preferably single fractions used. Preference may be given as peptide toxins also cell destructive acting snake venom such as the cobra snake venom Najatoxin-S can be used, each for loading the used for therapy dendritic cells. For the preparation of the pharmaceutical according to the invention effective fractions of the peptide toxins can be extracted from the poison cocktail, the over manual milking of the o.g. Animal species can be obtained, e.g. via column chromatographic Purification, specific toxin components (necrotic and cytotoxic acting peptide toxins) are selected. The analytics for differentiation the components contained in the fractions can be analyzed by HPLC-MS-MS (e.g. Device of Fa. Perkin-Elmer). It could be proven that it is a part of the high molecular weight substances on the Representation of toxic groups of the NX-NHX-NOX and SX type to peptide toxins is. This is confirmed by their mode of action in the cell experiment. (Burden R., Schrottenloher E., Weickmann D. (2000): In vitro experiments on Application / Use of cell-destroying poison components of the six-eyed crab spiders Sicarius spp. In biological cancer therapy.

Arachnologisches Magazine 2 (8) _ 1-7 and Lundblad R.L. (1995): Techniques in Protein Modification, CRC Press, London, Tokyo).

The according to the invention for the pharmaceutical effective loading substances used on natural Way to be won. These are of giant centenarians of the Genera Scolopendra hemiscolopendra, of serpents of the genera Bitis and Naja, of webspiders of the genera Loxosceles, Sicarius and pholcus and scorpions of the genus Parabuthus Poison, originally for Prey and pre-digestion of animal protein were developed. This natural Mode of action can be achieved by a preserving, gentle recovery of the toxicant base (e.g., by manual milking). Unlike traditional ones Milking of arthropods by means of an electrical method (Weickmann D. (1991): Attitude and toxicity of Sicaridae Scoreboard 16: 12-13; Weickmann D., Burda R. (1994): Electrophoresis of scorpion venoms, Electrophoresis Forum (1994), Abstracts, Techn. Universität München, Oct-24-26), in which the poison over the animals an electrical impulse which causes the contraction of the animals poison glands triggers, is withdrawn (the spiders are here preferably undercooled, scorpions and Skolopender give in to hypothermia hardly to no poison), according to the present Invention of poison cocktail over a manual procedure in which the animals are taking advantage of their natural Defense against the release of their poison can be stimulated.

According to one embodiment The invention provides a manual milking of the spiders. Thereby become genuine, unadulterated won native venom while in the electric milking of spider poisons by electron flow restructured substances or molecules are obtained which in changed their mode of action could be or substances in the poisons that may be present Otherwise the animal would not give up, while by the giant centipede through the conventional electrical method is dispensed no poison. The by electric Milking in addition substances released by the arachnids, but not necessarily the medical effectiveness of the individual contained in the total poison cocktail Negatively affect connections. By default, a quality control of the Raw poison over electrophoretic methods are carried out.

The following examples show advantageous embodiments of the invention, however, are not intended to limit the scope of the invention.

In The examples and the description will be made to the following figures Referenced:

1 shows SDS-PAGE of total venom cocktails from scorpions obtained in manual and electric milking; Lanes from left to right: molecular weight standard; Androctonus anst. Hector; mechanical milking, electric milking; Nebo hierichinticus, mechanical milking, electric milking; Opistophthalmus sp., Mechanical milking, electric milking.

Examples

1. Milking

a) Skolopender

to Manual milking animals are from a body length of about 10 cm of the genera Scolopendra and Hemiscolopendra, the Scolopendra and Hemiscolopendra Giant Caged Feuds Scolopendra morsitans, Scolopendra gigantea and Hemiscolopendra spp. used. The Skolopender with the fingers of a Hand gripped behind the head, with the palm of your hand fixed so that Do not use the animal with the last pair of legs transformed into tongs can sting. Then lets bite the animal through a foil stretched over a test tube is.

At the Bite is given at the same time poison, which is on the test tube collects. The poison thus collected is placed in a desiccator. This will then for at least 12 hours in a freezer at least minus Stored 14 degrees.

b) snakes

to Milking become snakes of the genera Bitis and Naja from a body length of about 50 cm used. Thereby the snakes with a firm grip fixed behind the head and the body is placed over the arm. Then you leave the snake through a slide that stretched over a beaker is, bite. Or you leave Carefully place the snake to be milked directly on the edge of a beaker or bite an evaporating dish. During the bite each poison is released, which is located on the bottom of the cup collects. The poison thus collected is dried in a desiccator. The dried poison can be at plus 4 degrees Celsius for several years be kept.

c) spinning

to Manual milking becomes adult females of the Loxosceles spider species Loxosceles laeta, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens, Loxosceles reclusa, Loxosceles deserta, in the area of Genus Sicarius the Sicarius spider species Sicarius hahni, Sicarius albospinosus, Sicarius gweni, Sicarius testaceus, in the field of Genus Pholcus the Pholcus spider species Pholcus phalangioides, Pholcus spp. (RSA), Pholcus spp. (Cuba) and Pholcus opilionides, with the fingers of one hand in supine position fixed, with a syringe in the other hand (2 ml Braun inject from B. Braun) with attached sterile Kneaf (20 or 21 by Becton Dickinson), the time of day did not matter, at a room temperature of 21 to 27 degrees Celsius, at a humidity level of about 60%, at the chelicerae by touching with the blunt side the cannula stimulated to deliver the poison. It is preferred that the Stimulation time no longer than 90 seconds, otherwise the animal will be unnecessarily stressed is suspended. After the poison drop appears on the poison claws this is over with the syringe the cannula reared. For Each animal will use a new syringe with a new cannula. Subsequently, will the cannula with the needle guard again locked. The closed syringe with wound poison is directly afterwards spent in a desiccator. This will then be in for 12 hours in mind a freezer kept at least minus 14 degrees Celsius

d) scorpions

to Manual milking will be animals of a body size of about 3 cm of scorpion species Parabuthus transvallicus, Parabuthus granulosus and Parabuthus villosus used. The scorpions at the Postabdomen are directly behind the poison bladder with a foam-coated tip Tweezers taken and the poison sting prefers a finely polished Aluminum plate (about 4 × 5 cm) contacted with a thickness of 2 mm. When contact the Scorpion drops a drop of poison on the aluminum plate, which then is spent in the desiccator. This will then be for at least 12 hours in a freezer kept at least minus 14 degrees.

2. Fractionation of the total poison

The frozen complete toxin is removed after removal from the freezer in 1 ml of solvent eg protein solvent from Carl Roth GmbH & Co KG (solvent for protein column chromatography: 9.25 M Tris / HCl, pH 6.5 to 7.3, 1, 92 M glycine, in distilled, deionized water (due to denaturation if no SDS is used in the buffer)). This will give poison in solution. The individual poison solutions of each species prepared in this way can be collected in a sterile, clean Teflon vial at room temperature. The sealed Teflon vial is then shaken on a vortex without foaming for 30 seconds to give a homogeneous solution. After mixing, the entire solution is passed through a Plexiglas funnel (to avoid contamination) into a standing transparent Plexiglas column having a diameter of 1.5 cm, a wall thickness of 2 mm and a height of 50 cm, and conical down to 1 , Open at 5 mm, filled with 20 ml gel (Sigma / Supelco, AcA 34; matrix 3% acrylamide 4% agroose; fractionation range (MW): proteins: 20 to 350 kDa; exclusion limit 750 kDa; bead diameters 60 to 140 Micrometer) introduced. The poison solution thus introduced passes through the gel and displaces the buffer in the gel. Upon complete penetration of the poison solution into the gel, an additional 150 ml of solvent (0.25 M Tris / HCl, pH 6.5 to 7.3, 1.92 M glycine) is added to the column. This additional solvent displaces as it passes through the gel the poison solution contained therein. The first 15 ml running down the column are residual buffers and are discarded. Depending on the animal species, up to 30 fractions of 4 ml each are collected after these 15 ml. The separation into 4 ml each is due to the physical and chemical properties of the individual fractions, detected by electrophoresis, preferably SDS-PAGE. Roti Load 1 + 2 (Carl Roth GmbH & Co., KG, Karlsruhe: SDS, glycerol, bromophenol blue, phosphate buffer, Roti Load 1 with mercaptoethanol, Roti Load 2 without mercaptoethanol) are used as the order buffer for peptide bond and protein protection. The individual fractions collected separately in sterile clean screwable 5 ml teflon vials. The quality control of the individual fractions is carried out by means of electrophoresis.

The The skin cell lysing activity of the fractions was determined by: the individual fractions from the total poison cocktail of the respective Animal species in experiment on human living cells in high to overdosed Quantities (about 200 to 500 μg / ml used medium) were tested. Application for loading dendritic Cells found those peptide toxins containing both healthy skin cells, prefers skin fibroblasts of different biology, as well degenerated skin cells, preferably 2 lines of malignant melanoma, destroyed. unconsidered remained those peptide toxins, which in addition to skin cells also more Cells (e.g., breast tissue cells and liver cells) damaged and sometimes even lysed. In the case of Sicarius, peptide toxins became in fractions 1-12 receive. These fractions contained peptide toxins in a molecular weight range from about 72 to about 168 kDa. A skin cell lysing, in particular malignant melanoma cell lysing peptide toxin is in fraction 10 contain.

For the pharmaceutically therapeutically effective loading of the dendritic cells according to the invention, for example, individual skin cell lysing fractions of the animal poisons are used. These fractions contained animal venom protein components in a molecular weight range of about 50 to 350 kDa or nonprotein toxins with at least one amido group in a molecular weight range of about 30-55 kDa. An SDS-PAGE of the total venom cocktail of scorpions is in 1 shown.

Around to clarify the structure of the individual substances, the respective Fractions e.g. over a HPLC-MS-MS and DAD-UV spectrometry (DAD or DADI: Direct Analysis of Daughter Ions, Direct Analysis of Daughter Ions). In the higher Molecular weight range from 10,000 could no known substances being represented. The skeletal provisions indicate an affiliation a part of the substances with at least one polypeptide type toxic components, ie polypeptide toxins. There were however also determined toxins with a molecular weight of 30-55 kDa, the no Have protein structure.

fractions with the same compositions collected together. For the further processing and storage become the individual fractions Freeze-dried, for example with the following parameters: The to lyophilizing fraction is in an open, with perforated Aluminum foil loosely covered Teflonvial, to minus 22 degrees Celsius cooled. For a safe freezing of the sample, a cooling time of 11 hours (at Skolopenders at least 20 hours). Then a vacuum of 0.200 mbar. After reaching the vacuum, the fraction heated to plus 4 degrees Celsius and for at least 24 hours while maintaining the vacuum maintained this temperature. After the lyophilization process is screwed the Teflonvial airtight with the lyopyhilisierten fraction. The storage time is at room temperature for about 3 months (for scolopopia toxins 4 weeks), at plus 7 degrees Celsius about 1 year (Skolopendergifte are at this Temperature is stable for about 5 months) and at minus 14 degrees Celsius about 5 years (Skolopendergifte should be stored for longer periods at minus 80 Degrees Celsius).

4. Loading dendritic cells with Petidtoxinen

3-5 ml of one Culture of dendritic cells (2.5 to 15 million dendritic Cells / 5 ml) prepared according to the above in this invention described methods were used with 0.5 to 2.5 ml of a skin cancer cell solution (supernatant a freshly tapped pure human skin cancer cell culture flask with about 250,000 Hauerkrebszellen / ml cell medium. Medium: 500 ml of DMEM-Ham's F-12, 10 ml of penicillin / streptomycin, 5 ml glutamine and 50 ml FBS). Subsequently, 1 to 2 ml of a Solution, the approximately 150 μg / ml Peptide toxi of fraction 3 from Loxosceles spiniceps were added. It was incubated for 24 hours at room temperature. Then it was under the sterile workbench with a previously autoclaved Pasteur pipette about 4 ml, medium supernatant (if possible without dendritic cells) and discarded. Then took place a further addition of 1 to 2 ml of the peptide toxin Lox.Tox. 3 (same concentration as first addition), so that a final concentration to peptide toxin of about 100 μg / ml present. Until the use of the thus loaded dendritic cells these are further incubated at 37 degrees Celsius in the incubator.

• X: viele eng beieinander liegende Banden auf dem Gel, und somit nicht eindeutig bestimmbar, ob Fraktion 5 oder 6.

The dendritic cells loaded with the toxins mentioned are collected / enriched, so that the most saturated solution of dendritic cells loaded with toxins arises. In the mixed tissue cell experiment, ie a culture of healthy tissue and skin tumor cells (cell culture: malignant melanoma: certainly as these identified cells, obtained by the patient after consultation with this, since 1995 also observed as long-term and subculture) were then the effects of different poisons, with which the dendritic cells are loaded, tested out. On average, the tumor cells (malignant melanoma cells) were 4 mm in diameter and about 1-2 mm thick. Loaded dendritic cells were added directly into the medium (500 DMEM-Ham's F-12, 10 ml glucamine, 50 ml glutamine and 50 ml FBS), although all tumor cell areas were attacked, but the procedure lasted the longest and repeated after 3 weeks had to become. Injecting dendritic cells into the tumor can be done very selectively, but many dendritic cells die off. The most effective method was to inject the cells loaded with the toxins directly to the edge of the tumor so that, by their nature, the dendritic cells can destroy the biologically active tumor border area first. The lysed tumor cells are digested (in part by macrophages). In all animal toxins tested, the lysed cell areas grew again with healthy cells. In this example, the effect of the following poisons was investigated:

• X: many closely spaced bands on the gel, and thus not clearly determinable, whether fraction 5 or 6.

The best and fastest tumor destruction after a single dose of loaded dendritic cells The tumor margin was with poisons of scolopenders, Bitis arietans and Loxosceles spiniceps achieved. This was a complete tumor destruction already after 6 hours with a single addition, with the lysed cell areas to grow with healthy cells.

If not all tumor cells destroyed are given a new gift of loaded dendritic every two days Add cells.

When solvent to the above active ingredients, the above-described solvent is preferably used.

Claims (39)

Pharmaceutical agent for the treatment of Tumors containing in a pharmaceutically effective amount: a) dendritic cells, which are loaded with b) toxins and / or optionally synergistically and / or antagonistically acting substances from the poison cocktail of scolopenders of the genus Scolopendra, and c) wherein as solvent in 15 ml 0.9 percent NaCl solution 7 ml of the homeopathic Substance Tarantula D4 can be used, and d) where to this Add up to 0.5 ml of a saturated solution of the entire poison cocktail be added by spiders of the species Loxosceles laeta, and e) depending on the required proportions of the total poison cocktail in anhydrous formic acid which in turn is 1 to 2 ml of the total extract of Poisons of ants bulldog ants are added, taking care of these Amount refers to a quantity of the total poison cocktails of 10 ml. Pharmaceutical agent for the treatment of tumors, containing in a pharmaceutically effective amount: a) dendritic cells, these are loaded with b) toxins and / or optionally this synergistic and / or antagonistic substances from the poison cocktail of scolopendons of the genus Scolopendra, and c) being as solvent in 15 ml of 0.9 percent NaCl solution 7 ml of the homeopathic Substance Tarantula D4 can be used, and d) where to this Add up to 0.5 ml of a saturated solution of the entire poison cocktail be added by spiders of the species Loxosceles gaucho, and e) depending on the required proportions of the total poison cocktail in anhydrous formic acid which in turn is 1 to 2 ml of the total extract of Poisons of ants bulldog ants are added, taking care of these Amount refers to a quantity of the total poison cocktails of 10 ml. Pharmaceutical agent for the treatment of tumors, containing in a pharmaceutically effective amount: a) dendritic cells, these are loaded with b) toxins and / or optionally this synergistic and / or antagonistic substances from the poison cocktail of scolopendons of the genus Scolopendra, and c) being as solvent in 15 ml of 0.9 percent NaCl solution 7 ml of the homeopathic Substance Tarantula D4 can be used, and d) where to this Add up to 0.5 ml of a saturated solution of the entire poison cocktail be added by spiders of the species Loxosceles Mallorca, and e) depending on the required proportions of the total poison cocktail in anhydrous formic acid which in turn is 1 to 2 ml of the total extract of Poisons of ants bulldog ants are added, taking care of these Amount refers to a quantity of the total poison cocktails of 10 ml. Pharmaceutical agent for the treatment of tumors, containing in a pharmaceutically effective amount: a) dendritic cells, these are loaded with b) toxins and / or optionally this synergistic and / or antagonistic substances from the poison cocktail of scolopendons of the genus Scolopendra, and c) being as solvent in 15 ml of 0.9 percent NaCl solution 7 ml of the homeopathic Substance Tarantula D4 can be used, and d) where to this Add up to 0.5 ml of a saturated solution of the entire poison cocktail be added by spiders of the species Loxosceles Menorca, and e) depending on the required proportions of the total poison cocktail in anhydrous formic acid which in turn is 1 to 2 ml of the total extract of Poisons of ants bulldog ants are added, taking care of these Amount refers to a quantity of the total poison cocktails of 10 ml. Pharmaceutical active ingredient according to one of claims 1 to 4, characterized in that, instead of the feature b), the toxins and / or optionally synergistically and / or antagonistically acting substances from the poison cocktail of scolopenders of the genus Hemiscolopendra be used. Pharmaceutical active ingredient according to one of claims 1 to 4, characterized in that, instead of the feature b), the toxins and / or optionally synergistically and / or antagonistically acting substances from the poison cocktail of snakes of the genus Bitis be used. Pharmaceutical active ingredient according to one of claims 1 to 4, characterized in that, instead of the feature b), the toxins and / or optionally synergistically and / or antagonistically acting substances from the poison cocktail of snakes of the genus Naja be used. Pharmaceutical active ingredient according to one of claims 1 to 4, characterized in that, instead of the feature b), the toxins and / or optionally synergistically and / or antagonistically acting substances from the poison cocktail of spiders of the genus Loxosceles be used. Pharmaceutical active ingredient according to one of claims 1 to 4, characterized in that, instead of the feature b), the toxins and / or optionally synergistically and / or antagonistically active substances from the poison cocktail of spiders of the genus Sicarius be used. Pharmaceutical active ingredient according to one of claims 1 to 4, characterized in that, instead of the feature b), the toxins and / or optionally synergistically and / or antagonistically acting substances from the poison cocktail of spiders of the genus Pholcus be used. Pharmaceutical active ingredient according to one of claims 1 to 4, characterized in that, instead of the feature b), the toxins and / or optionally synergistically and / or antagonistically acting substances from the poison cocktail of scorpions of the genus Parabuthus can be used. Pharmaceutical active ingredient according to one of claims 1 to 4, characterized in that, instead of the feature b), the toxins and / or optionally synergistically and / or antagonistically acting substances from a combination of the poison cocktails one or several of the animal species Scolopendra, Hemiscolopendra, Bitis, Well, Leoxosceles, Sicarius, Pholcus or Parabuthus are derived. Pharmaceutical active ingredient according to one of claims 1 to 4, characterized in that the substances are selected from toxins of the scolopender species Scolopendra morsitans or Scolopendra gigantea, as well as combinations of several of these toxins. Pharmaceutical agent according to claim 5, characterized in that that the substances are selected are obtained from Hemiskolopender species Hemiscolopendra sp. Pharmaceutical agent according to claim 6, characterized in that that the substances are selected are made from toxins of the snake species Bitis arietans (Puffotter), Bitis gabonica (Gabunotter), Bitis nasicornis (Rhino viper), Bitis atropos, bitis caudalis, or bitis peringueyi, as well as combinations of several of these toxins. Pharmaceutical agent according to claim 7, characterized in that that the substances are selected are derived from the toxins of the spas Naja melanoleuca, Naja pallida, Well, well sputatrix, as well as combinations of several of these Toxins. Pharmaceutical agent according to claim 8, characterized in that that the substances are selected are made from toxins of the araneomorphic recluse species Loxosceles laeta, Loxosceles spiniceps, Loxosceles bergeri, Loxosceles parami, Loxosceles rufescens, Loxosceles reclusa or Loxosceles deserta, as well as combinations of several of these toxins. Pharmaceutical agent according to claim 9, characterized in that that the substances are selected are made from toxins of the six-eyed crab spider species Sicarius hahni, Sicarius albospinosus, Sicarius testaceus or Sicarius argentinensis, as well as combinations of several of these toxins. Pharmaceutical agent according to claim 10, characterized in that the substances are selected from toxins of the dormouse species Pholcus phalangioides, Pholcus opilionides, Pholcus spp. (RSA), or Pholcus spp. (Cuba), as well as combinations of several of these toxins. Pharmaceutical agent according to claim 10, characterized in that that the substances are selected are derived from toxins of the thick-tailed scorpion species Parabuthus transvallicus, Parabuthus granulosus or Parabuthus villosus, as well as combinations of several of these toxins. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the dendritic cells are of human origin are. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the toxins by fractionation of the poison cocktail of the mentioned species were obtained. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that by fractionation of the poison cocktail gained toxins along with more, in each fractions present substances are used without another Purification takes place. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the toxins are used in pure form. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the toxins are present in recombinant form. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the toxins are peptide toxins. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the peptide toxins have a molecular weight of approx. 50-350 kDa have. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the peptide toxins are necrotic, cytotoxic and / or have apoptotic properties. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the dendritic cells interact with the toxins Lipofektion and / or laser microinjection are loaded. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the toxins have an amido group, but no protein structure and have a molecular weight of 30-55 kDa. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the dendritic cells in a therapeutic effective amount are loaded with the toxins, the dendritic Cells are brought into contact with a medium for loading, in which the toxin concentration in the range of 0.5-250μg / ml, preferably 80-250μg / ml, still more preferably about 170 μg / ml, lies. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that synthetically or recombinantly produced peptide toxins be used. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that derivatives of the peptide toxins are used, one or more amino acids added, deleted and / or have been replaced, with the toxic properties the toxin remain substantially retained. Pharmaceutical active ingredient according to one of the preceding claims, characterized characterized in that the dendritic cells continue to interact with interferons, Cytokines, cytokine receptors, anaphylatoxins, glycosides and / or antibodies are loaded. Process for the preparation of a pharmaceutical active substance for loading dendritic cells according to one of the preceding Claims, characterized in that the dendritic cells with a or more toxins as defined in claims 1-34 be brought to associate the cells with the toxins. A method according to claim 35, characterized in that the Loading the dendritic cells with the toxic substances incubated become. Method according to one of claims 35 or 36, characterized that the toxic substances are absorbed into the dendritic cells or associate with the dendritic cells. Method according to one of claims 35 to 37, characterized that the loading by lipofection and / or laser microinjection the toxic substances take place. Method according to one of claims 35 to 38, characterized that the pharmaceutical agent for the treatment of tumor diseases, especially of tumors of the skin and the mucosa is suitable.