WO2005027942A1

WO2005027942A1 - Pharmaceutical active ingredient made from snake poison to fight against melanomas

Info

Publication number: WO2005027942A1
Application number: PCT/DE2004/002058
Authority: WO
Inventors: Dirk Weickmann
Original assignee: Toximed Gmbh
Priority date: 2003-09-16
Filing date: 2004-09-15
Publication date: 2005-03-31
Also published as: DE112004002332D2; DE10342747A1

Abstract

The invention relates to pharmaceutical active ingredients used in the treatment of melanomas by combining substances obtained from biogenic poisons and/or oral secretions, said secretions being recovered from certain types of vipers.

Description

Active pharmaceutical ingredient against melanoma

Everyone is responsible for the ingestion of food for the preservation of life

Living beings rely on the range of plants and animals that can be reached. But not everything is safe to eat here.

Many plants and animals use to protect their own life and for their own food, on their special organism and its special

Needs coordinated, so-called biogenic, poisons. These biogenic poisons have found their place in the interplay of different types of life in the course of long development periods.

That is why every adult wild animal still recognizes dangerous plants and poisonous animals in its natural environment.

Plants or animals can have a primarily toxic effect through the production of toxins or can only acquire secondary toxicity by ingesting toxic substances from the living or inanimate environment.

The use of these biogenic poisons began in human history as long ago as they were used to kill prey with poisoned weapons.

From the beginning, however, there were certain precautions to use these poisons safely

Basic knowledge of their treatment and effectiveness is required.

The experiments carried out further, the composition of the chemical

Decoding the build-up of biogenic poisons later led to the targeted search for certain active substances as the actual cause of the effects observed.

In particular, after the demand made by Paracelsus (1493 - 1541) to isolate the active substances from medicinal plants, which contributed to the development of latrochemistry, that is, chemistry with regard to its medical field of application, these efforts should have intensified. Above all, the art of distilling fabrics has been put to the service of research and has provided a variety of essential oils and volatiles. But the methods known at that time were insufficient for the isolation of other active substances or even for their chemical breakdown. It was not until the beginning of the 19th century that the development of technical skills in chemistry had advanced far enough to usher in the era of isolating pure active substances from biological material. First, the differences in the solubility of the investigated substances in different solvents were used to separate the active substances from the accompanying substances. The differences in the distribution behavior between two immiscible liquid phases, in the volatility and in the chemical reactivity were observed, for example with precipitation agents.

The development of chromatographic processes in the middle of the 20th century made possible a huge boom in separation technology, the way to identify active substances for combating diseases. Based on the distribution between a mobile and a stationary liquid phase, on the adsorption, the molecular sieve effects, the ion exchange, the affinity (especially of proteins) for certain chemical compounds (e.g. enzyme substrates) and the mobility of charged molecules in the electric field, a large number became new separation techniques developed

Tumors, the most dangerous and feared diseases of our time, are currently being combated in a very radical and less environmentally friendly way. The following can be used as simple, characteristic keywords: steel, radiation and chemotherapy.

On the one hand, this means that, if reasonably attainable, tumors are in principle cut out with the steel of a knife, burned by a wide range of radiation, or destroyed by so-called chemotherapy with aggressive cytostatics that also attack healthy cells. A spatial limitation of the operating area is not possible with normal treatments with a scalpel or with ionizing radiation. Healthy body cells are inevitably destroyed. The undesirable side effects of chemotherapy are well known. In contrast, however, cancer therapy that deserves its name was also attempted in a more subtle way. For this purpose, the rich treasure of nature was used. For this purpose, among other things, many highly effective substances in therapeutic doses isolated from toxic organisms are used as medicinal substances A pharmaceutical active ingredient is known from DE 199 61 141 A1, in which it has been found that components of the spider venom from spiders of the Sicaridae family can be used for the treatment of tumor diseases. Mainly, a peptide toxin from the poison of this type of spider, a further antagonistic substance obtained from the poison and / or a combination of these components are used medically. This active ingredient can be used for the treatment of tumor diseases and in parallel or in support of tumor operations and residual tumor tissue can be destroyed. In therapy, genetically modified body cells (tumor cells) can be destroyed, since the active ingredient in question recognizes the changed surface structure of such cells and kills them without complications. cannot be used pharmaceutically.

However, this known active ingredient does not act in combination against melanomas in vivo.

Malignant melanoma is a malignant tumor that originates from the pigment cells of the skin. The most important risk factors for developing melanoma are a healthy skin type and exposure to UV rays, whereby sunburns in childhood are of particular importance. The warning symptoms of liver spots include an increase in size, a change in color, itching, easy vulnerability with a tendency to bleed, as well as an irregular limitation of the pigmented skin area. Since the chances of a cure are only good with early diagnosis and therapy, people at risk should be examined by a dermatologist once a year. The first step in the treatment is surgical removal of the melanoma, and further therapy depends on the stage of the disease.

Around eight out of a hundred thousand people develop malignant melanoma. Light-skinned people with inadequate tanning tendencies are particularly affected. Melanoma develops preferentially on skin areas that are exposed to the sun, such as the face, neck, arms and lower legs. About 60% of melanomas develop from liver spots (nevi), which have often been unremarkable for years.

The number of diseases among the black population is only one sixth of that of the fair-skinned population. In the black population especially the less pigmented parts of the body such as mucous membranes, as well as hand and

Infested foot surfaces.

In terms of appearance, four main types of melanoma can be distinguished: the superficial spreading melanoma, the nodular melanoma, the

Lentigo - Maligna - Melanoma and Acral - Lentigindous Melanoma.

The superficial spreading melanoma is the most common type of melanoma with 70%. It grows only on the surface over a long period of time and forms flat, irregularly shaped stoves with different colors from black, gray, blue and brown to white. Sometimes it turns into a nodular shape after years, combined with vertical growth.

The frequency of nodular melanoma is approximately 15%. It bulges hemispherically beyond the skin level and is from gray-brown to reddish - blue-black

Colour. Because of its rapid vertical growth, its healing prognosis is particularly poor.

Acral - nodular melanoma is present in about 7% of the cases. It mostly appears on the palms of the hands and feet and in the nail area. It is in the

Early stage around brown to brown-black spots that later for wetting and

Bleeding tends.

Lentigo - malignant melanoma is less common in about 5% of cases.

Older people in particular are affected on the face. The initially flat, later knotty herds are irregular and out of focus. The color is brown, occasionally with black spots.

Amelanotic melanoma is rare. It is not colored and therefore particularly difficult to see.

Malignant melanoma starts from the pigment-forming cells (melanocytes) that lie on the epidermis. The tumor cells grow both in the horizontal direction and in the vertical direction. The horizontal growth leads to a visible increase in the size of the melanoma on the skin. The vertical growth is important for the forecast. The basement membrane as the lowest layer of the epidermis temporarily represents a certain barrier to the blood and lymphatic vessels of the epidermis. If it is penetrated, the cancer cells can spread throughout the body via the blood and lymph channels, attach to other organs and multiply there. This scattering, also known as metastasis, may have already taken place in the case of very small tumors.

If you suspect melanoma, the suspicious skin must always be removed completely by surgery, taking at least three centimeters of healthy skin on the outside. The skin is then examined under the microscope (histology). The tumor thickness and the tumor penetration depth are the most important prognostic factors in the assessment. The entire body must be examined to determine the extent of the tumor. X-rays, sonography and computed tomography are mostly used. Infection of the lymph nodes or other organs can be demonstrated.

It is the object of the active ingredient according to the invention to cause tumor cells in melanomas to be killed without complications.

This object is achieved by an active ingredient which has the features of one of the independent claims 1 to 4 and is produced by a method according to claim 23.

Active substances from the poison and oral secretions of vipers, namely the following types, are preferably used: a) Vipera berus, b) Vipera wagneri, c) Vipera ursini, and d) Vipera aspis.

Generally, the snakes are divided into two main groups. 1. Vipers (Viperidae) or otters and 2. Poisonous snakes (Elapidae). The group of vipers is divided into two subgroups, the real vipers, which is why

Vipera berus (adder) and the Vipera aspis (Juraviper) belong to, and the

Pit vipers.

43 types of real vipers are known. They are spread across Europe, Asia and Africa and are related to the American pit vipers

(Crotalidae), which also include the rattlesnakes.

Viperidae differ from pit vipers by the absence of the "pit," a sensory organ in front of the eyes.

All vipers have a typical venomous apparatus that is more effective than most other snakes. Each of the two poison glands opens directly at the bottom of a long hollow poison tooth. When the snake strikes, the fangs enter the prey and the poison emerges from the tip of the fangs. In this way it is very effectively injected deep into the victim. The fangs are also mobile ..

When in use, they straighten up almost perpendicular to the upper jaw, while they are otherwise folded back, enclosed in a soft connective tissue sheath, lying on the roof of the mouth.

The European vipers have a rather strong body with a short tail and often a triangular head. The back scales are strongly keeled and often almost all signs on the top of the head are divided. The relatively small eyes have vertical pupils. In contrast to most other snakes, the after shield is undivided among the vipers. With some species it comes down to the

Snout tip to form a soft, scaly snout horn.

Vipers often live on the ground, although some species can climb stone piles and low bushes. They rarely swim. All species are active during the day when the night temperatures are high enough, even at night. The food consists mainly of small mammals (mice, voles, shrews), birds or lizards.

Unless they are disturbed, all European vipers move relatively slowly. They often hunt out of their hiding place by thrusting at the prey that passes by to inflict the poison bite on them, mammals are not caught after the bite. Rather, they are left to the poisonous effect and then sought out and devoured by the viper by tracing the scent trail. In contrast, birds, and lizards Insects are usually held and the gobble begins as soon as they stop moving. Vipers can also hunt for prey in the corridors of mammals.

Vipera berus is also known as adder. It is a small, relatively slender venomous snake in which the elongated head is only slightly separated from the body. It grows to a size of 60 cm to 85 cm in the wild. The copper-red iris is characteristic of the adder. The basic color of her body is variable. The color palette ranges from beige and all shades of brown to yellow and gray to reddish. On the back there is usually an uninterrupted zigzag band which stands out from the basic color and whose appearance is a distinguishing feature for the different subspecies. The snake's body sides are adorned with a series of round spots at regular intervals. The basic color of the male animals is always much lighter than that of the female animals. Rare red-brown adores without a back label are rare.

Vipera wagneri, also called Wagner's Bergotter, comes from northeastern Turkey and Azerbaijan. It is a 70 cm to 90 cm long, muscular snake and has a strikingly short tail.

The body color of the male animals is light gray, while the female animals are colored light brown. There are ocher-colored spots on the back that touch in the middle of the back. These have a dark brown or black border and are separated from each other by light crossbeams. The tip of the snout is lighter in the female animals than in the male animals. Wagner's mountain otters like to stay close to the water, especially on small streams or rivulets. Depending on the season, they are day or night active. These animals are actually very shy, but when they are in danger they hiss loudly, raise their front bodies at an angle and bite.

Vipera ursini are found in southern locations up to an altitude of 3000 meters. These snakes prefer the steppes and meadows. In principle, they inhabit the ground, but can also climb well. Most of these animals are active during the day. They reach a body length of 35 cm to 50 cm and show similarities Adder on. The oval head is relatively little separated from the body. The snout is narrower and more pointed than the adder. The eyes have vertically slit pupils. Strongly keeled back scales give a roughened overall impression at this point.

Vipera aspis, also known as aspis viper, is also known as the actual European venomous snake because it only occurs here. It is 60 cm to 70 cm long and has a typical square head, with the body narrowing sharply towards the neck. The body coloration is very variable and can be stone gray, brown, reddish brown, brick red and orange. Vipera aspis has a typical viper-like stocky body. However, the male animals are longer and slimmer than their female counterparts and have a dark back pattern, consisting of mutually transverse spots that can merge into a zigzag or wavy band. The eyes have vertically slit pupils in bright light,

According to the invention, in addition to the peptide toxins, further enzymes of the vipers in question are used in the types mentioned under a) to d), and further biogenic substances are used in the oral secretion.

It must be ensured that the substances obtained have a certain molecular weight, which can be 28 kDa, 32 kDa or 35 kDa.

The active ingredient according to the invention can optionally contain an antagonistic or synergistic and / or penetrating substance from the total poison cocktail or oral secretion of the relevant animal species to the respective peptide toxin or oral secretion.

The antagonistic or synergistic substance is preferably a phospholipase or a hyaluronidase or a combination of both substances.

It is further preferred that the antagonistic or synergistic substance is a mixture of the phospholipases and hyaluronidases and / or toxins present in other types of animals. The peptide toxin and the substance which is antagonistic and / or synergistically active are preferably obtained from the viper total poison cocktail by a fractionation process, and it is further preferred that the pharmaceutical active substance contains a peptide toxin and a substance which has an antagonistic or synergistic effect which consists of different fractions. As a result, the effect of the pharmaceutical active ingredient can advantageously be tailored to the type and / or size of tumor to be treated.

The same applies to the substances obtained from the oral secretions.

The peptide toxin, or the oral secretion, and the substance having an antagonistic and / or synergistic effect can be obtained by known fractionation processes for the separation of proteins from the total poison raw mixture or the mouth saliva. It is preferred that the peptide toxin and the substance having an antagonistic or synergistic effect thereof are obtained by gel chromatography, HPC, affinity chromatography and / or ion exchange chromatography.

It is also preferred that the peptide toxin is present as an active pharmaceutical ingredient in such an amount that the active ingredient has a destructive effect on tumor cells.

Furthermore, the required proportions are chosen so that the active ingredient according to the invention has no or only a slight toxic effect in the patient to be treated. Of course, the amounts of the active pharmaceutical ingredients must also be matched to the type of tumor to be treated and the physical, possibly also psychological, circumstances of the respective patient. The preliminary tests required for such a coordination are to be carried out by the person skilled in the art in the context of animal tests and / or ethically justifiable tests on the patient on the basis of his specialist knowledge and ability. Also preferred is a pharmaceutical active ingredient in which the amount of peptide toxin or oral secretion and the substance having an antagonistic or synergistic effect thereon comprises an amount of peptide toxin or oral secretion and antagonistic or synergistic substance which depends on the one to be treated Tumor is chosen.

It is further preferred that the pharmaceutical active ingredient according to the invention contains conventional carriers and auxiliary substances, such as antibiotics, antifungals, antituberculotics, agents against parasites, cytostatics, amino acids, enzymes which promote wound healing and / or mitotic inhibitors. Preferred are penicillin / streptomycin, polymyxin / genetic mycin (5%), mitopodocide, vinca rosea alkaloids, bromelaina or bromelains.

In the pharmaceutical active ingredient according to the invention, the peptide toxin or the oral secretion and the antagonistic or synergistic substance are used in combination. However, it is also possible to use the individual substances in active pharmaceutical ingredients and to make the special effects of the substances available for therapeutic use.

It is also possible to produce the described active ingredients chemically and synthetically or by recombinant means using genetic engineering methods. As is usual with chemical substances, the present invention also includes derivatives and salts of the substances provided according to the invention. For example, the peptide toxin can comprise one or more additions, substitutions and / or deletions of amino acids, it naturally having to be ensured that the medicinal effect according to the invention is retained.

The active ingredient described is obtained by methods customary in chemical process engineering. These include in particular fractionation processes; however, other methods can also be used, for example immunological methods, in order to get the desired substances out of the total poison cocktail or mouth secretion A preferred process for the preparation of an active ingredient according to the invention, comprising at least one peptide toxin and / or at least one substance having an antagonistic or synergistic effect, at least one peptide toxin and / or at least one antagonistic or synergistic substance from the poison of animals of the above under a) to d) mentioned species, has the following steps:

Obtaining a raw poison mixture or oral secretion by methods known per se, and fractionation of the mixture in order to obtain the peptide toxin and the substance having an antagonistic or synergistic effect in fractions which are as separate as possible from one another. Optional combination of different fractions with one another or with poisons originating from other organisms or with antagonistic or synergistic substances in order to obtain a pharmaceutically active substance.

The poisons in question contain various peptide toxins and various substances which have an antagonistic or synergistic effect thereon, and other active substances which are also of medical and therapeutic relevance. All of these substances can be used therapeutically in a specific ratio, to be determined by the person skilled in the art, in a medical active substance.

It is pointed out that the fractionation process shows, by way of example only, a possibility of obtaining the peptide toxins and the substances having an antagonistic or synergistic effect. Further configurations are possible.

The same applies to the extraction of oral secretions.

It is preferred that the raw poison mixture used, or the. Mouth saliva, is obtained from female animals of the type mentioned.

The process for obtaining poison and oral secretions from the vipers mentioned under a) to d) is carried out for the animals in a species-appropriate and gentle manner in the following form: a) the viper is fixed and a hand is held behind the head, b) with the other hand approach the viper with a sterile sponge, c) the sponge is held upright and the viper is brought closer, d) at a certain distance the viper bites into the sponge and you pull yourself with the sponge back from the viper.

The substances obtained are removed from the sponge, collected and then cleaned.

In addition to peptide toxins, enzymes and mucopolysaccharides are also obtained.

It is important here that only substances are used to produce the active substance according to the invention which have a molecular weight that is 28 kDa, 32 kDa or 35 kDa.

In the method according to the invention, it is also preferred that the raw poison mixture or the oral secretion is homogenized before the fractionation, and it is further preferred that the fractions are deep-frozen before further processing and more preferably lyophilized.

Surprisingly, combinations of peptide toxins or oral secretions contained in the animal venom and counter-directed (for this purpose antagonistic or synergistic) enzymes or peptide toxins in combination with oral secretions, in appropriate concentrations and proportions for the treatment of tumor diseases and in parallel or in support of tumor operations and (residual) tumor tissue is destroyed. Here, at least one peptide toxin or at least one substance acting antagonistically or synergistically comes from the poison of animals of the species mentioned.

For example, according to the invention, the destruction of tumor tissue not recorded during the operation and the prevention of local tumor metastasis in the organism can be achieved. During therapy, genetically defective body cells (tumor cells) can be destroyed on the one hand, because the Surface protein structure of such cells is changed and the phospholipases used according to the invention can recognize or selectively bind and lyse these tumor cells which have changed their surface structure. On the other hand, tissue in desired, locally delimited areas - here tumor tissue-predestined tissue areas - can be killed without complications. The mode of operation is based on the native, mutually influencing modes of action of the peptide toxins and the substances present in the animal poison, which have an antagonistic or synergistic effect, as follows:

Phospholipases hyaluronidases are also described as so-called penetrating enzymes. It is the case that the enzymes mentioned make tissue more permeable to the active ingredient according to the invention via digestive functions. In addition, they can recognize genetically defective body cells (tumor cells) and these themselves or by infiltration of necrotic or cytotoxic peptides coupled to them are killing. In this invention, antagonistic or synergistic substances are understood to mean, for example, phospholipases and hyaluronidases or peptide toxins from animals of the aforementioned genera, although it is not excluded that other antagonistic or synergistic substances are present in the animal venom, which can also be used according to the invention. Human phospholipases, especially type A phospholipases, would also be conceivable in an autologous blood preparation as a therapy for precursor cancer cells (genetically defective but divisible cells). However, the immune status is so poor that the amount of these phospholipases is not equivalent to the amount of genetically defective cells (Bisswanger H. (1994): Enzymkinetik. VCH Weinheim; Zollner H. (1993): Handbook of Enzyme Inhibitors, Part A. 271 272 and 383-388, VCH Weinheim; Zollner H. (1993): Handbook of Enzyme Inhibitors, Part B, VCH Weinheim).

In order to prevent unwanted cell destruction, according to the invention, depending on the type and size of the tumor to be treated, a comparison with respect to absolute and relative amounts of the components of the active ingredient according to the invention can be carried out in vitro on living human cells (healthy and tumorous). of the tissue type to be treated. Attention to the tendency to spread is of the greatest importance. This can be clarified in preliminary tests in comparison of the tumor tissue strength to the tissue surrounding the tumor.

The mode of action of the total poison cocktail, or individual substances separated therefrom by column chromatography and characterized by the molecular weight, can be carried out by testing them in corresponding healthy and tumorous human cell lines.

According to the present invention, the peptide toxins preferably originate from the same organism as the substances which have an antagonistic or synergistic effect thereon and / or further active substances optionally present. In this way, the effective interaction or counterplay of these substances developed by nature can be exploited.

The pharmaceutical active substances according to the invention can be produced in such a way that first a crude poison mixture is obtained by methods known per se and fractionation of the crude poison mixture is carried out by fractionation methods, likewise known per se, for the separation of proteins. This serves the purpose of obtaining the peptide toxins and the substances which act antagonistically or synergistically in a form which is as separate as possible from one another or in separate fractions. Subsequently, different fractions can be combined to produce a pharmaceutical active ingredient, or individual fractions can be combined with peptide toxins originating from other organisms or substances having an antagonistic or synergistic effect. Individual fractions can also be used to produce an active pharmaceutical ingredient. Hyaluronidases from snake venoms, for example from cobra venoms, can preferably be used as antagonistic substances. This can be combined with one or more fractions from substances obtained from vipers of the types mentioned under a) to d). It is also possible according to the invention to combine the fractions with other suitable active ingredients and / or with carriers and auxiliaries customary in pharmacy for the production of pharmaceutical active ingredients. To produce the active pharmaceutical ingredients according to the invention, specific poison components (necrotic and cytotoxic peptide toxins) and natural substances having an antagonistic effect (stop substances of the phospholipase and hyaluronidase type) can be selected from the animal venom, for example by purification by column chromatography.

Production of active substances according to the invention

To obtain the biogenic active substances, subadult females of the above-mentioned types a) to d) were stimulated to release the poison. The room temperature was usually about 21 to 27 degrees, the air humidity 50% to 70%. The time of day didn't matter.

A new syringe with a new cannula was used for each animal. The cannula was then closed again with the cannula protective cover. The sealed syringe and the drawn-up poison were then immediately placed in a desiccator. This was then kept in a freezer at at least 14 degrees Celsius for at least 12 hours. In the syringe with the frozen total poison, the cannula protective cover was removed after removal from the freezer. The cannula was placed in solvent, e.g. Protein solvent (solvent for protein column chromatography: 0.25 M Tris / HCL, pH 6.5 to 7.3, 1, 92 M glycine, in distilled, deionized water) and immersed in 1 ml. No SDS is used in the buffer due to denaturation, which resulted in poison in solution. The cannula was then removed. The individual poison solutions in syringes (5 pieces) prepared in this way were collected by disengaging (squirting) in a sterile, clean Teflon vial at room temperature. The sealed teflon vial was then shaken on a vortex without foaming for 30 seconds to obtain a homogeneous solution.

After mixing, the entire solution was placed in a standing transparent plexiglass column using a perspex funnel (to avoid contamination) had an inner diameter of 1.5 cm, a wall thickness of 2 mm and a height of 50 cm and was tapered at the bottom to 1.5 mm, was filled with 20 ml of gel. (Details: Aca 34; Matrx 3% Acrylamide 4% Agarose; Fractionation range (MW): Proteins: 20 to 350 kDa; Exclusion limit: 750 kDa; Bead diameter: 60 - 140 micrometers). The poison solution thus introduced ran through the gel and displaced the buffer contained in the gel. After the poison solution had completely penetrated the gel, an additional 165 ml of solvent (0.25M Tris / HCl, pH 6.5 to 7.3, 1, 92 M glycine) were applied to the column. This additional solvent displaces the poison solution contained in it as it passes through the gel. The first 15 ml that ran out of the bottom of the column was residual buffer and was discarded. After these 15 ml, 40 fractions of 4 ml each were collected. The separation into 4 ml was due to the physical and chemical properties of the individual fractions, demonstrated by electrophoresis, preferably SDS-PAGE. Rot Load 1 + 2 (Carl Roth GmbH & Co KG, Karlsruhe: SDS, glycerol, bromophenol blue, phosphate buffer, Roi Load 1 with mercaptoethanol, Roti Load 2 without mercaptoethanol) was used as the application buffer for peptide binding and protein protection. The individual fractions were collected separately in sterile, screwable 5 ml Teflon vials. The quality control of the individual fractions was carried out by means of electrophoresis.

In order to clarify the structure of the substances, the individual fractions were examined using HPLC-MS-MS and DAD-UV / - spectrometry (DAD or DADI: direct analysis of daughter ions, direct analysis of daughter ions). Known substances could not be shown in the higher molecular weight range (from 10,000 Da). However, the basic framework determinations indicate that the substances belong to the polypeptide type with toxic components (= polypeptide toxins) and, on the other hand, to the phospholipase and hyaluronidase type.

Fractions with the same composition can be collected together. The individual fractions were freeze-dried for further processing and storage, for example with the following parameters:

The fraction to be lyophilized was cooled in an open teflon vial loosely covered with perforated aluminum foil to minus 22 degrees Celsius. to A freezing time of the sample allowed a cooling time of 11 hours. Then a vacuum of 0.200 mbar was applied. After the vacuum had been reached, the fraction was heated to plus 4 degrees Celsius and kept at this temperature for at least 24 hours while maintaining the vacuum. After the freeze-drying process, the teflon vial was screwed airtight with the lyophilized fraction. The storage time is approx. 6 months at room temperature, approx. 1 year at plus 7 degrees Celsius and approx. 15 years at minus 14 degrees Celsius.

Tumor cells - destructive effect of the active substances according to the invention

It is currently not possible to offer comprehensive preventive therapy due to the diverse types of pathogenesis of tumor diseases. For locally diagnosed tumors (primary or subsequent), surgical removal comes first. A problem that arises here is metastasis if tumor tissue that has not been completely removed comes into contact with air when it is cut through. This problem can be solved by applying pharmaceutical active substances according to the invention to the cut surfaces during surgical tumor removal. Since the operation does not bring the desired results in the vast majority of cases, the use of the active pharmaceutical ingredients according to the invention is to be regarded as a therapy which is gentle on the patient. Positive results were shown in so-called permissible medical attempts at healing in patients who had undergone therapy. At the beginning of the test series, the fresh or dissolved total poison cocktail obtained and its effect depending on the concentration on different Zeil lines were tested. A satisfactory result could not be achieved, since the poison always killed the entire cell cultures, the time required for the killing decreasing with increasing concentration of the poison used.

During lengthy and detailed trials, it was surprisingly found that certain toxins in the active ingredient according to the invention have special lysing effects, in accordance with their molecular weights.

Claims

Expectations

Claim 1: Pharmaceutical active ingredient for the treatment of: Melanomas containing in a pharmaceutically effective amount: a) at least one peptide toxin, and b) at least one oral secretion, and c) at least one enzyme, and d) at least one substance which acts antagonistically to it or acts synergistically Substance, with at least the peptide toxin, or at least the oral secretion, or at least the enzyme and optionally the antagonistic or synergistic substance and / or the penetrating substance from the poison, or. the oral secretion, derived from vipers of the type Vipera berus and where e) these substances each have a molecular weight which is 28 kDa, 32 kDa, or 35 kDa.

Claim 2: Pharmaceutical active substance for the treatment of: Melanomas containing in a pharmaceutically effective amount: a) at least one peptide toxin, and b) at least one oral secretion, and c) at least one enzyme, and d) at least one substance which acts antagonistically or acts synergistically Substance, with at least the peptide toxin, or at least the oral secretion, or at least the enzyme and optionally the antagonistic or synergistic substance and / or the penetrating substance from the poison, or. the oral secretion, derived from vipers of the type Vipera wagneri and where e) these substances each have a molecular weight which is 28 kDa, 32 kDa, or 35 kDa. Claim 3: Pharmaceutical active substance for the treatment of: Melanomas containing in a pharmaceutically effective amount: a) at least one peptide toxin, and b) at least one oral secretion, and c) at least one enzyme, and d) at least one substance which acts antagonistically to it or acts synergistically Substance, with at least the peptide toxin, or at least the oral secretion, or at least the enzyme and optionally the antagonistic or synergistic substance and / or the penetrating substance from the poison, or. the oral secretion, from vipers of the type Vipera ursini and where e) these substances each have a molecular weight which is 28 kDa, 32 kDa, or 35 kDa.

Claim 4: Pharmaceutical active ingredient for the treatment of: Melanomas containing in a pharmaceutically effective amount: a) at least one peptide toxin, and b) at least one oral secretion, and c) at least one enzyme, and d) at least one substance which acts antagonistically to it or acts synergistically Substance, with at least the peptide toxin, or at least the oral secretion, or at least the enzyme and optionally the antagonistic or synergistic substance and / or the penetrating substance from the poison, or. the oral secretion, derived from vipers of the type Vipera aspis and where e) these substances each have a molecular weight which is 28 kDa, 32 kDa, or 35 kDa. Claim 5: Active pharmaceutical ingredient according to one of claims 1 to 4, characterized in that peptide toxins, enzymes and / or mucopolysaccharides are obtained from the poison and / or the oral secretion of the vipers in question.

Claim 6: Active pharmaceutical ingredient according to one of the preceding claims, characterized in that for the treatment of melanoma in a pharmaceutically effective amount a peptide toxin is added, which is obtained from the venom of rattlesnakes of the Crotalus family.

Claim 7: Active pharmaceutical ingredient according to one of the preceding claims, characterized in that a peptide toxin is added in combination for the treatment of melanoma in a pharmaceutically effective amount, which is obtained from the poison of scorpions of the Androctonus bicolor family.

Claim 8: active pharmaceutical ingredient according to one of the preceding claims, characterized in that the antagonistically active substance or the synergistically active substance and / or the penetrating substance originate from a different organism than from one of those under a) to d) and in claims 6 or 7 types mentioned. Claim 9: Pharmaceutical active substance according to one of the preceding claims, characterized in that the antagonistically active substance or the synergistically active substance and / or the penetrating substance is a phospholipase or a hyaluronidase or a combination of both substances.

Claim 10: active pharmaceutical ingredient according to one of claims 4 to 4, characterized in that the peptide toxin and the substance having an antagonistic effect thereon, or the substance having a synergistic effect and / or the penetrating substance, was obtained from the respective total poison cocktail by a fractionation process.

Claim 11: Active pharmaceutical ingredient according to one of claims 1 to 4, characterized in that the mucopolysaccharides and the substance having an antagonistic effect thereon, or the substance having a synergistic effect and / or the penetrating substance, were obtained from the respective oral secretions by a fractionation process.

Claim 12: Pharmaceutical active substance according to one of Claims 1 to 4, characterized in that the peptide toxin and the substance having an antagonistic effect thereon, or the substance having a synergistic action and / or the penetrating substance, are characterized by gel chromatography, HPLC, affinity chromatography and / or ion exchange chromatography the respective total poison cocktail was obtained. Claim 13: active pharmaceutical ingredient according to one of claims 4 to 4, characterized in that the mucopolysaccharides and the substance having an antagonistic effect thereon, or the substance having a synergistic effect and / or the penetrating substance by gel chromatography, HPLC, affinity chromatography and / or ion exchange chromatography from the respective oral secretions were obtained.

Claim 14: Active pharmaceutical ingredient according to one of Claims 1 to 4, characterized in that the peptide toxin and the antagonistically active substance or the synergistically active substance and / or the penetrating substance was obtained as follows: by using a column with an inner diameter of 1, 5 cm and a height of 50 cm, which tapers to 1.5 mm at the bottom and which contains 20 ml of an AcA 34 same chromatographic gel, matrix: 3% acrylamide 4% agarose, fractionation range 20 to 350 kDa, exclusion limit 750 kDa, bead diameter 60 - 140 µm is filled. The corresponding poison was applied to the gel in a homogeneous state in 0.25M Tris / HCl, pH 6.5 to 7.3, and 1.92M glycine in distilled, deionized water. When the poison solution has passed through the gel and 165 ml of a solution of 0.25 M Tris / HCl, pH 6.5 to 7.3, and 1.92 M glycine in distilled, deionized water have been applied and the first 15 ml of the run discarded and collected in 4 ml fractions, the peptide toxins are in fractions 1, 2, 4, 7, 9 and 10 and the antagonistic substances or the synergistic substances and / or the penetrating substances are in fractions 3.5 , 6,8,11 and 12 ..

Claim 15: active pharmaceutical ingredient according to one of claims 1 to 4, characterized in that the mucopolysaccharides and the antagonistic substance, or the synergistic substance and / or the penetrating substance was obtained as follows: by using a column with an inner diameter of 1.5 cm and a height of 50 cm, which is conical down to 1 , 5 mm and which is filled with 20 ml of a chromatographic gel AcA 34, matrix: 3% acrylamide 4% agarose, fractionation range 20 to 350 kDa, exclusion limit 750 kDa, bead diameter 60 - 140 µm. The corresponding oral secretions in 0.25M Tris / HCl, pH 6.5 to 7.3, and 1.92M glycine in distilled, deionized water were applied to the gel in a homogeneous state. When the oral secretion solution has passed through the gel and 165 ml of a solution of 0.25 M Tris / HCl, pH 6.5 to 7.3, and 1.92 M glycine in distilled, deionized water have been applied and the first 15 ml of the run discarded and collected in 4 ml fractions, the peptide toxins are in fractions 1, 2, 4, 7, 9 and 10 and the antagonistic substances or the synergistic substances and / or the penetrating substances are in fractions 3.5 , 6,8,11 and 12 ..

Claim 16: active pharmaceutical ingredient according to one of claims 1 to 4, characterized in that it contains a peptide toxin and a substance having an antagonistic effect thereon, or a substance having a synergistic effect, which originate from different fractions of the respective total poison cocktail.

Claim 17: Pharmaceutical active substance according to one of claims 1 to 4, characterized in that it contains a mucopolysaccharide and a substance having an antagonistic effect thereon, or a substance having a synergistic effect, which originate from different fractions of the respective oral secretions. Claim 18: active pharmaceutical ingredient according to one of the preceding claims, characterized in that the amount is selected so that a spatial and / or temporally controlled spread in the tissue is ensured.

Claim 19: Pharmaceutical active substance according to one of the preceding claims, characterized in that the amount of peptide toxin, as well as oral secretion, and antagonistically active substance or synergistically active substance and / or penetrating substance is selected depending on the tumor to be treated.

Claim 20: active pharmaceutical ingredient according to one of the preceding claims, characterized in that it contains conventional carriers and auxiliary substances and / or further active substances.

Claim 21: Pharmaceutical active substance according to one of the preceding claims, characterized in that it is isotonic solutions, protein solutions, amino acid solutions and / or germicidal solutions, preferably Ringer's solution, 0.9% NaCl solution, human albumin solution, as usual carriers and auxiliaries and / or glutamine solution, and that it contains antibiotics, antifungals, antituberculotics, agents against parasites, amino acids, enzymes which promote wound treatment, mitotic inhibitors as further active ingredients and / or contains cytostatics.

Claim 22: Pharmaceutical active substance according to one of the preceding claims, characterized in that a derivative of the peptide toxin, or of the oral secretion, or of the enzyme, and / or the antagonistically active substance or the synergistically active substance and / or the transit substance in the Active ingredient is contained and / or the peptide toxin, or the oral secretion, or the enzyme, and / or the substance having an antagonistic effect or the synergistic substance and / or the penetrating substance is produced chemically-synthetically or by recombinant biotechnological methods and they correspond in their effect to the active substances contained in the total poison cocktail, or the oral secretion, of the respective animals or the substances having an antagonistic or synergistic effect and / or penetrating substances and derivatives thereof.

Claim 23: Process for the preparation of a pharmaceutical active substance according to one of the preceding claims, comprising in a pharmaceutically effective amount at least one peptide toxin or an oral secretion, and at least one substance which acts antagonistically or synergistically and / or at least one penetrating substance, wherein the peptide toxin or the oral secretion from animals according to one of claims 1) to 4) and optionally the substance having an antagonistic effect or the synergistic substance and / or the penetrating substance from the poison or the oral secretion of animals according to one of claims 1 ) to 4), with the following steps: Obtaining oral secretions, animals, and obtaining a total poison raw mixture by methods known per se and fractionating the mixture around the peptide toxin and, optionally, the substance having an antagonistic or synergistic effect and / or the penetrating substance and optionally further substances in fractions which are separated from one another if possible to obtain; Combination of different fractions of the peptide toxin with fractions which contain antagonistic or synergistic substances and / or penetrating substances for this purpose, or with antagonistic or synergistic substances and / or penetrating substances originating from other organisms, in order to obtain a pharmaceutically active substance.

Claim 24: Method according to Claim 23: characterized in that the total poison raw mixture is preferably obtained from female animals in each case.

Claim 25: Method according to one of claims 23 to 24, characterized in that the total poison raw mixture is homogenized before the fractionation.

Claim 26: Method according to one of claims 23 to 25,: characterized in that the fraction is deep-frozen before further processing to a pharmaceutically active substance and more preferably lyophilized. Claim 27: Method according to one of claims 23 to 26, characterized in that the method for obtaining poison and oral secretions from the vipers mentioned in claims 1 to 4 is carried out in the following manner: a) the viper is fixed and a hand behind its head held, b) with the other hand, the viper is approached with a sterile sponge, c) the sponge is held upright and the viper is approximated, d) at a certain distance the viper bites into the sponge and one pulls with the Swam back from the viper. e) the substances obtained are removed from the sponge, collected and cleaned. f) only substances are then used whose molecular weight is 28 kDa, 32 kDa or 35 kDa.