Patent Application
20110038846
The invention relates to a pharmaceutical composition, the use of the pharmaceutical composition to treat the brain tumor, manufacturing processes, and a Kit of parts containing the pharmaceutical composition.
Occur annually in Germany around 7000 cases of serious brain tumor. Besides this is about an astrocytoma with II and III malignancy grade, as well as IV grade glioblastoma and multiforme glioblastoma. Brain tumor is often diagnosed as a secondary result. Often occur in patients bleedings in the cervical and skull territory, which is associated with loss of consciousness and/or severe pain, partially very strong. Thus is really necessary the admission into the hospital.
Brain tumor treatment is presented very cumbersome. In particular to the emergence of the glioblastoma often the forecast is not positive for patients. By removing the tumor with surgery can be alleviated the symptoms and the duration of life may be extended with only a few months. A sustainable recovery is very rare.
Currently against tumors, the most dangerous and most feared disease of our era, is struggling in a very radically way, without protecting the patient. To this purpose we present a few simple keywords: steel, rays and chemotherapy. This means once, that the tumor, if accessible, is removed in principle with the bistoury, is destroyed by a radiation on a flat surface, or by a so-called chemotherapy, which through the aggressive cytostatic affects also the healthy cells. Both through the normal treatment with the bistoury, that by the ionisating radiation, it is not possible the territorial delimitation of the operation area. Inevitably are destroyed also the healthy cells of the body. The unwanted side effects of the chemotherapy are generally known, as the disadvantages of therapy with rays.
In particular the removal of the brain tumor by surgery has however side effects. Is often required a second operation of the patient. At the first intervention and the removal of the brain tumor is produced a large cicatrized area. There, in addition to the healthy glial cells can be stored also diseased glial cells, thus malignant. By are often cells derived from the tumor and generate metastases. With this is scheduled a diffuse recurrence. This can not be treated by the traditional methods of treatment. Therefore are often fatal to the patients.
After the surgery follows usually a therapy with rays and/or chemotherapy. In the chemotherapy is typically used Cisplatin in various combinations. Since the beginning of 2002 is used also the medicine with oral administration, with the name of Temozolomid, from Essex Company, as the product's name being Temodal®. And Glivec® is also a cytostatic that is used. In addition to the known side effects, such as vomiting, feeling sick, depressions, by the cancer and the nerves affections caused by chemotherapy, to the products mentioned above appears also the problem that in order to treat the brain tumors, these must pass the blood barrier from the brain. Usually the active substances pass the blood barrier from the brain in only 0.4 percent up to 5%. The composition and the functioning way of the blood barrier of the brain are understood only in primary way. This barrier is intended to protect the brain from harmful substances, on the other hand, however, it allows a limited input power.
Our body is composed of organic individual systems and organs, which require for their operation different conditions but constant, such as alimentation substances, hormones or electrolytes. All the organs are related by the blood circulation. Because in the blood are found all the components needed for the nourishment, but also for the elimination of the toxins from the body, the filtering systems are careful to allow the passage only of the substances necessary to certain organic systems. In this report we know the blood barrier of the tissues, also called the blood barrier of the parenchyma, the blood barrier of the liver, the blood barrier of the liquor, the blood barrier of the brain, the liquor barrier of the brain, the blood barrier of the nerves, the blood barrier of the retina and the barrier of the placenta.
To this filtering mechanism, through the so-called barrier effect is restricted to prevent the passage in the organs system of certain substances in the blood stream, in the case in which the components are not necessary or are required in a small amount for these organs systems.
These “barriers” are not independent organs, but are composed of a series of cells and spaces between the cells, allowing the passage of the blood, of the nutrient substances and of certain chemicals or retain them as endotelic macromolecules. May have the effect of membrane of the lipids in the wall of the vessels, at the passage of the substances which do not dissolve the lipids or with selective effect of the active process of transportation in the capillaries. The brain and the nervous tissue are protected by two systems of filtration, the blood barrier of the liquor and of the blood barrier of the brain.
The existence and the functioning of the so-called barrier blood of the brain is already known for more than 100 years and has been demonstrated through experiment by Paul Ehrlich in 1885 already. In the central nervous system, the spaces between the neurons are almost totally filled by glial cells and their terminations. The entire metabolism of the nervous cells is carried out by these glial and endotelic cells. They contribute to the formation of the nerves cells and of the nerves containers, as well as to their alimentation and isolation. A form of the glial cells are the astrocytes. They have many extensions, with which are held to the wall of the capillaries and constitute an endotelic surface, surrounded on all sides, but without descents. These endotelic cells are chained by relation elements, “tight junctions” and provided with a selective possibility of passage of the substances, allowing the passage of the particles with the diameter less than 20 mm. In this way, the general metabolism is conducted through this endotelic netting, which allows the passage according to the need of the substances in the blood like a biological filter, but maintains to the distance from the nervous system the substances that are dangerous for the functioning of the brain.
The endotelic knitting and the endotelic cells, that are surrounding the capillaries like a basal membrane bears the name of blood barrier of the brain. Are allowed to pass without restrictions the acidic substances, carbon dioxide Ide, glucose-D hexose-D, certain amino acids-L and substances necessary to the nutrition of the brain. In the same way are handed the products that are decomposed in the blood. The final registration final of the astrocytes constitutes a certain barrier for certain hormones, substances which do not dissolve the lipids, which dissolve the water and the chemicals, such as proteins and ensure by this the maintaining of a constant milieu for the nervous system neurons.
The astrocytes cell structure is so ordered, that it constitutes an obstacle to the substances with increased molecules and of the organisms. However under normal conditions is not fully thick, so certain particles can always pass through this barrier. In the case of the infections, of the traumas, inflammations, poisoning, hypodixose, fever and in tumors field, the close links, those tight junctions between the endotelic cells are dilated by the inflammation of the astrocytes and become significantly more permeable to other substances. The modification of the distance is the consequence of the swelling and of the deflation of the endotelic cells. Neither the basal membrane of the capillaries is not a closed layer. Depending on the containers network density, are constituted pores in the membrane, which participate actively in the change of the substances.
Already long before the possibility of the treatment with antibiotics has increased the capacity of crossing the blood barrier of the brain by the artificial producing of the fever, similar to the infections process, for the therapy of the central nervous system syphilis and for the shock therapy in psychiatry, so arised the possibility to be administrated medicines directly on the brain. After the end of the conditions that influence the blood barrier of the brain, is brought back the temporarily permeability.
For the chemotherapy of the patients with brain metastasis of the solid tumors are investigating in the meantime Termozolomid (Termodal®), a lipofil alkalic. It weakens the blood barrier of the brain and increases the tumor sensitivity to radiation, in case of simultaneous radiotherapy.
In contrary is researched also the possibility of conducting the brain therapy in a more subtle way, based on natural products.
In this respect, among others, are used many substances extracted from poisonous beings, with increased effect of drugs in therapeutic doses.
The use of these biological poisons has already begun early in the human history. To safely use these poisons were necessary at the outset basic knowledge regarding the treatment and their effect. Further research relating to deciphering the chemical composition of these biological poisons later led by the specific investigation of certain active substances to the proper use of the substances which have provoked the effects studied.
The powerful booming effusion of the separation technique, the path to the ascertaining of the active substances to fight against the diseases is constituted by the discovery of the chromatographic process in the middle of the 20th century. Starting from the division between a mobile phase and a stationary liquid, from the absorption, from the effect of sieve of the molecules, from the change of ions, affinitated (especially of the proteins) to the chemical relations defined (eg enzymes substrate) and the mobility of the molecules in the electric field, has developed a great number of new techniques for separation.
Just in the new time have developed many active pharmaceutical substances from the biological poison.
This is known from the PCT/EP00/19202 an active pharmaceutical substance, to which it has been found that certain components of the venom of the spiders in the sicis used a peptide toxin extracted from the poison of this species of spiders, then the substance extracted from the poison, with antagonistic effect and/or the combination of these components is used medically. Is recommended the use of the peptide toxin for example from Sicarius species for treating the breast carcinoma, lung carcinoma, adenocarcinoma, liver carcinoma, as well as the melanoma. Is not mentioned the possibility of treating the brain tumors, such as astrocytoma and the glioblastoma. In consequent way is not discussed either the problem of passing the blood barrier of the brain.
The active substances described in PCT/EP00/19202 may be used to treat the disease with tumors, as well as in parallel or as a support in the operations with tumors and the debris of tissues from tumors. In therapy can be destroyed the cells from the body genetically modified (tumor cells), whereas the active substance recognizes the modified structure of the surface of these cells and it eliminates them without complications. The total poison of these species of spiders, a so-called cocktail of the different substances can not be used from pharmaceutical point of view because of the fatal effect, even in small doses.
This known active substance however it does not have effect in vivo, and in any case to a special type of the brain tumor, such as a oligodendroglioma or oligodendrocytoma. For a successful therapy is needed for this active substance to pass in a large quantity from the blood barrier of the brain.
Another combination of poisonous substances is published in PCT/EP2006/063281. The published combination of the various poisonous substances partially neutralized each other, thus the destruction effect of the tumor cells is reached in unsatisfactory way.
It was found surprisingly that the peptide toxin from the Loxosceles unbinds the peptide toxin of the Latrodectus, so the peptide toxin from the Loxosceles passes in an insignificant quantity passing through the blood barrier of the brain. On an electrophore canvas after the mixing of the peptide toxin of the Latrodectus and of the Loxosceles it has not shown a distinct fascia, that unfortunately a scratch, caused by the peptide pieces disposed from the peptide toxin of the Latrodectus. The spiders of the Loxoceles species belong, as well as the Sicarius species to the family of the Sicarides. Based on a small degree of tight relational of the two species of spiders can not be expected any effect of the mixture of the peptide toxin of the Sicarius species with the one of the Latrodectus species.
Therefore because of the present invention is the production of a composition that by the effective passing as possible of the blood barrier of the brain to have as effect the destruction without complications of the body cancer cells, from the field of the brain tissue, especially of the rare brain tumor, of the oligodendroglioma.
Another task of the present invention is also the achievement of a manufacturing procedure of one pharmaceutical composition for treating the brain tumor.
Another task of the present invention is also the production of a combination of active substances, which facilitate an effective treatment of the brain tumor.
The tasks will be solved by the independent criteria signs. The main developments and variations of the invention are found in the additional criteria.
A first aspect of the present invention it refers to a pharmaceutical composition for treating the brain tumor that contains in an efficient quantity from pharmaceutical point of view:
The peptide toxin of the spiders from the Sicarius species and/or the saliva of the vipers from the Elaphe species are able to destroy the cells of a tumor. By combination with the poison of the spiders in the Latrodectus species, the poison that destroys the tumor is transported through the brain blood barrier into the brain and there it that can fight against the tumor. The poison of the spiders in the Latrodectus species has so an effect of substance that passes through the blood barrier of the brain. For the first time this combination makes it possible to destroy the tissues of the tumors in the brain. Unexpectedly the peptide toxins from the Latrodectus were not disposed of by the peptide toxins from the Sicarius, so the peptide toxins from Latrodectus have acted as transfer substances for the other toxins that are tumor cell destroyers and have passed the blood barrier of the brain, then by contact with the peptide toxins from Sicarius and/or Elaphe could destroy the tumors in the brain field, for example the oligodendrocytes.
Subsequently it was found surprisingly that the peptide toxins from the Elaphe are also able that in contact with the peptide toxins from the Latrodectus to pass the blood barrier of the brain and on the other hand to kill the cells astrocytomatic as the glioblastomatic cells. In this case, the peptide toxins were extracted from the saliva of the Elaphen species. A combination of the peptide toxins of the Elaphen species with the peptide toxins from the Loxosceles on the contrary had not any success. The peptide toxins of the Elaphen species, extracted from the saliva of the ones from the Elaphen, destroyed the peptide particles from the active substance of the Loxoceles, so it could not demonstrate an effect later. But the combination of the peptide toxins from the Sicarius and the species of Elaphe had effect and it could pass, even if with the help of the peptide toxins from the Latrodectus, through the blood barrier of the brain.
The peptide toxins extraction can be achieved by known methods. Here a reference is made to the content described in PCT/EP00/12902. Through various chromatographic procedures, for example HPLC techniques certain mixtures of poisons can be totally divided in individual fractions and then the fractions can be studied in terms of their effect. The tumorous cells destruction effect of certain fractions of the peptide toxins can be defined in experiments performed with the help of the cell cultures. The effect of the factions from the peptide toxins that destroy the tumorous cells in contact with the substance of crossing the blood barrier of the brain, extracted from the Latrodectus can be then experimented by models of animals known. Examples in this regard are found in PCT/EP00/12902, to which here is made a total reference to the content and whose content was taken entirely in the registration. As described in PCT/EP00/12902 through the peptide toxins from the Sicarius, the specialist can isolate from the peptide toxins of the Elaphe saliva of and the total poison cocktail of the spiders in the Latrodectus species the particles with effect and to test them in the way presented above. Here reference is made to PCT/EP2006/063281, whose content was taken entirely in this recording and to which the author makes reference. This presentation shows the use of the peptide toxins from the Latrodectus, which are able to pass through the blood barrier of the brain and simultaneously shows the ability to let to pass through the blood barrier of the brain also other peptide toxins, and namely those of Sicarius and Elaphe.
In Latrodectus can, alternatively at the fractions of peptide toxins, interfere also with the total poison of the spiders. In all cases it is very important that the Latrotoxin to be totally removed from the poison cocktail, because the pharmaceutical composition, which contains as a means of crossing through the blood barrier of the brain the total poison or part of the spiders poison in the Latrodectus species, to be able to be used without being a danger for the patients. In this time are known the Latrotoxins alpha and gamma. These must be removed at least to such an extent, that the toxic side effects can be excluded in the case of accumulating in patients. For the pharmaceutical composition are preferred those fractions from the total poison cocktail from the Latrodectus, which contain the active substances for the passing through the blood barrier of the brain, respectively that make possible the passage of the anti-tumorous active substances from Sicarius and Elaphe from this barrier.
According to a preferred development of the present invention, the pharmaceutical composition contains additionally Parapoxvirus Ovis inactive, in particular of D1701 provenance. Especially are preferred here the commercially available preparations having the name of Zylexis®, from the company Pfizer®. Here it is about Parapoxvirus Ovis inactivated, of D170 provenance, to which it has been additionally added polygeline and Aqua ad injection.
1 ml of Zylexis® resuspended contains:
According to a preferred development of the present invention, the spiders of the Sicarius species will be selected, being composed of the subspecies Sicarius oweni, Sicarius testaceus, Sicarius hahni, Sicarius albospinosus, Sicarius brasiliensis, Sicarius terrosus and Sicarius terrosus formae.
The spiders of the Latrodectus and Sicarius species and preferred milked, in order to take possession of the total poison cocktail:
The spiders are milked mechanically, without the electrical stimulation of the poison glands, at temperatures between 21 and 27° C. The milking process is accomplished in a sterile space, under a stereo magnifying glass; through the magnifying glass is observed if the spiders bite in the capillaries, it is important that they do not tears them up, because the digestive enzymes can destroy the peptide toxins. The poison cocktail thus obtained is decomposed into component parts through a basic chromatographic process and/or other typical one.
In example 2 is described in more detail the extraction of the peptide toxins from the Sicarius. The specificities of the peptide toxins extraction from the Sicarius, which are applicable to those of Elaphe and Latrodectus, are described in PCT/EP00/12902, so the experts can obtain based on this stage of the technique without any problems, fractions of the spiders poison.
According to another preferred development of the present invention, the peptide toxin of the spiders in the Sicarius species, is the peptide toxin HT1 and/or HT2. In example 2 is described in detail the way of obtaining this peptide toxin.
According to another preferred development of the present invention, the composition contains additionally also Lachesis D6. Lachesis D6 (Lachesis means snake) of DHU (German homeopathic Union) is preferred as a mean of dissolution for the peptide toxin from the Sicarius. In particular at the production of the composition it can be used preferably Lachesis D6.
According to another preferred development of the present invention, the spiders of the Latrodectus species are selected from the group, being composed of the subspecies Latrodectus bishopi, Latrodectus curacaviensis, Latrodectus dahli, Latrodectus erythromelas, Latrodectus hasselti, Latrodectus katipo, Latrodectus menavodi, Latrodectus rhodesiensis, Latrodectus revivensis, Latrodectus schuchi, Latrodectus tredecimguttatus, Latrodectus variolus, Latrodectus hesperus, Latrodectus corallinus, Latrodectus geometricus and Latrodectus obscurior.
According to another preferred development of the present invention from the species of the Latrodectus spiders are preferred the subspecies Latrodectus geometricus and Latrodectus obscurior.
To another development of the present invention it can be studied the mix of the poison of the Sicarius or Latrodectus species. The mixture may be of the different types of Sicarius or Latrodectus species or also the mixture of different populations of a type.
The second aspect of the present invention it relates to the use of pharmaceutical composition, as the one described above, to produce a medicine to treat the brain tumor and the glioblastoma, particularly to treat an oligodendroglioma.
Through the combination of poison or poison part of the animals in the Sicarius species or the saliva of the Elaphe species with poison or part of poison of the animals in the species Latrodectus, corresponding to the invention, it can be passed through the blood barrier of the brain. The poison of the Latrodectus animals serves besides this as a passage substance, which facilitates a passing through the barrier.
Another aspect of the present invention, refers to the process of making the composition, having the following steps:
According to a preferred development of this aspect before mixing it will be added an additional immunomodulator, preferably Parapoxvirus ovis, especially of provenance D1701.
According to a preferred development of this aspect is dissolved at least one peptide toxin Sicarius in the Lachesis D6, before adding to the solution of NaCl, are preferred both peptide toxin-Sicarius.
According to a preferred development of this aspect, the first toxin of peptide-Sicarius is a peptide toxin-Sicarius HT1 and/or the second peptide toxin-Sicarius will be a peptide toxin-Sicarius HT2.
Subsequently are preferred as passing substances the pharmaceutical preparations of phospholipase of the types of Sicarius, which facilitate the taking of the peptide toxin by the tumorous cells. The substances of this type and their extraction are exemplary described in PCT/EP00/12902.
Another aspect of the present discovery relates to a kit of parts for treating the brain tumors and the glioblastoma, comprising a pharmaceutical composition, as the ones described above and an incense extract and/or bamboo leaves.
The main components of the incense are the resins in which are found boswelia acids and essential oils. In addition to the boswelia acids, which belong to the pentacicilic Triterpins group, in the resin is also found a tetraciclic Triterpin, the acid of tirucal.
In the present discovery is used mainly the H15 Ayurmedica preparation. Here it is about the tablets, which contain a standard level of 400 mg of dry extract of boswelia cerata. The dosage is regulated depending on the need. It may differ widely, situating between the values of 3 tablets every week up to 25 tablets daily, preferably being of 10 tablets weekly up to 20 tablets daily.
The incense extract is used as an alternative to the Cortisone, in order to prevent the brain edemas in the case of the treatments with the appropriate pharmaceutical composition of the present discovery. The edemas appear in a large number in the case of the treatment of the brain tumors, especially when they are destroyed very quickly, this means the cases where they diminish very quickly.
In the followings will be presented in detail the various poisonous substances.
The tumorous cells are destroyed by using the peptide toxins, which are extracted from the poison of the animals in the Sicarius species, spiders with six eyes. Are preferred the subspecies Sicarius oweni, Sicarius testaceus, Sicarius hahni, Sicarius albospinosus, Sicarius argentinensis, Sicarius brasiliensis, Sicarius terrosus and Sicarius terrosus formae for extracting the poison. The most preferred peptide toxin is the one extracted from the subspecies Sicarius oweni, Sicarius testaceus, Sicarius argentinensis, Sicarius terrosus and Sicarius terrosus formae, where the last two subspecies are the most preferred.
The peptide toxins can be extracted by means of separation known, such as for example by electrophoresis with gel or the chromatography, especially base chromatography. The extraction of the primary poison cocktail from spiders is described below.
Alternatively or additionally it can be used the saliva of the vipers from the Elaphe species. The saliva is extracted by keeping a rodlet of cotton in front of the snake, in which eventually the snake may bite. The saliva remained on the cotton rodlet will dissolve from the rodlet with the help of a means of dissolution, such as Lachesis D6 or a solution of 0.9% of NaCl. The resulting solution can be restrained by the procedures already known.
For the passage through the blood barrier of the brain, are preferred the active substances from the poison of the spiders in the Latrodectus species (“black widow”). These belong to the species of the spiders with sphere (Theridiiade). The characteristic of these animals is the large back side, reminiscent of a sphere. The front side of the body is very small comparing with the cesta. Males reach a length of 5 mm without feet, and the females mostly between 10 and 18 mm. These animals have a brownish color to shiny black. Almost always are found signs of red color, orange or yellow on the back side of the body. They can be found on the most various territories, rarely are found also in the cities. The forest is not preferred in the majority as a life space.
Are advantaged in terms of the use the following subspecies:
The last two subspecies are preferred for the crossing through the blood barrier of the brain especially in the case in which the patients who are treated are children.
For the composition according to the present discovery are particularly preferred the subspecies Latrodectus geometricus and Latrodectus obscurior.
The Black Widow, as it is known in homeopathy, is found mainly in America, along the Pacific Coast, to Canada. The poison homeopathically prepared from the black widow is an important substance for the treatment of the Angina pectoris. This spider it usually maintains away from the popular localities and it lives near the ground, among stones and brushwood. However, where a spider is found, usually there are several copies in the environment.
Latrodectus bishopi lives in central and southern Florida.
Latrodectus curacaviensis lives in the area of North America and South America, and among the Netherlands Antilles. The females are on the whole body of shining black color, only on the abdomen have striking signs of red or orange color. The frightful legendary poison is quite dangerous for people, but in rare cases it is also lethal.
Latrodectus dahli is found in Iran.
Latrodectus erythromelas lives in Sri Lanka.
Latrodectus hasselti is encountered from India to the New Zealand and is often found in basements, garages, barns and esterios WC's. These spiders live with pleasure also under the batches of wood or under other construction materials or debris.
Latrodectus katipo is the only poisonous spider in the New-Zealand. It lives mostly in the artificial environs of New-Zealand, except for the extreme South. Is found near the soil, in the grass or cut wood. The bite of the Katipo spider can be deadly, especially as the antidote has its effect in three days.
Latrodectus menavodi is found in Madagascar and Seychellen.
Latrodectus rhodesiensis lives in southern Africa.
Latrodectus revivensis lives in Israel, especially in the Negev.
Latrodectus schuchi (newly Latrodectus lilianae) lives in Europe only in Spain, specifically in Aragon.
Latrodectus tredezimguttatus is found in southern Europe very often on the fields of grain, before all on the territory between the sea, partly also in Asia and Africa. The appearance of these spiders is characterized by a body entirely glossy black and 13 red spots on the back side. The net wove often irregularly is often found in nearby the soil. Because to this net are always wove new and new stitches, it is stable enough. The center of the net forms a basket shaped net and often is found under an object, such as a rock, a branch or even the crack of a wall.
Latrodectus variolus is found for example in America in Michigan. The female specimens have a red dot on the inferior part of their posterior, the males do not have this feature, but they wear a red and yellow stripe on the back.
The poison of Latrodectus is composed of 6 to 7 proteins, with the molecular weight from 5 kDa up to 130 kDa, of which the Latrotoxin is a neurotoxin with an increased effect. Is used the total of the poison cocktail or part of the poison of the Latrodectus species referred to in paragraphs a) to n), and in particular the subspecies Latrodectus geometricus and Latrodectus obscurior. The Latrodectus species referred to in paragraphs m) and n) can be used also for children of 12 years old.
The substances used according to the discovery for the pharmaceutical composition, may be obtained naturally from animals. These poisons are usually developed for attachments and previous digestion of the animal proteins. This method of natural action can be achieved by obtaining the poisonous substance by the protective extracting, with the maintenance of the function (for example by manual milking).
In contrary to the traditional methods of milking from the anthropodes, using an electrical method (Weickmann D. (1991): The attitude and the poison of the Sicariidaes. Arachronologic Index 16, p. 12-13; then Weickamnn D., Burda R (1994): Electrophoresis of scorpion venoms. Electrophoresis Forum 1994, Abstracts, Technical University of Munich), in which the poison is obtained from animals with an electrical impulse, that causes the contraction of the venomous glands of animals (animals are here preferably cooled), after the present discovery, the cocktail of venom is obtained through a manual method, by which the animals are stimulated by using their natural behavior of defense to release the poison.
According to a formula for the presentation of the discovery is provided a way of manually milking the spiders. By this it will be obtained genuine venom, unadulterated, while for example by the electrical milking method, through the flow of electrons were retained unstructured substances, respectively molecules, which could modify their effect, or in the poison could be present also substances which under other conditions the animal din not release them. These substances may, but do not need to necessarily influence adversely the efficiency of the medical composition contained in the poison cocktail. By electrophoretic process can be checked the quality and/or it can be analyzed from the point of view of the standard the mixture of primary poison.
On manual milking will be used primarily the underadult females from the species and subspecies above mentioned, these being fixed with the finger of a hand, in the position on the back and by tapping with the part of the trunk of a sterile cannula positioned on a sterile injection, of the maxillaries, are stimulated to release the venom. The space temperature must be between 21 and 27 degrees, air moisture of 50% up to 70%. Has no role the day time.
It must be taken into consideration that the stimulation time must not last more than 90 seconds, otherwise the animal will be exposed to an additional stress. After the apparition of the poison drops on the maxillaries with poison, it will be draw with the help of the cannula into the injection. Finally the cannula will be closed with cannula's protector. Closed the injection, together with the poison collected will be at the end brought directly in an exsiccator.
It is possible that the active substances described, which are found in various animal poison, to be produced by chemical-sunthetical methods or by gentechnological methods in recombined form. How it is the habit in the case of the chemical substances, the present discovery includes also derivatives and salts of the substances corresponding to the discovery. For example, the peptide toxin may contain one or more substitutes and/or deletions of amino acids, where is needed to be ensured that it will be maintained the medical effect corresponding to the discovery. The extraction of the active substances described takes place by the traditional methods of the technique of the chemical procedures.
The active substances of the present discovery will be used in the form of such a pharmaceutical composition, in which they will be mixed with carriers or carrier materials in doses, so that the disease to be treated or at least mitigated. Such a composition can contain (in addition to the active materials and to the carriers), dilution substances, filling materials, salts, tampons, stabilizers, solubilisation means and other materials well known in the technique. The term “pharmaceutical tolerable” defines a non-toxic material, that does not disturb the biological activity of the substances contained inactive, respectively of the active substances. The selection of the carriers depends on the method of treatment.
The pharmaceutical composition may further comprise other materials that increase the activity of the active substance or complements its activity or its use in the treatment. Such complementary factors and/or means may be contained in the pharmaceutical composition, which can achieve a synergistic effect and can minimize the side effects or the undesirable effects.
The formulation techniques, respectively of preparation and administration of the compounds of the present registration can be found in “Remington's is Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pa., latest edition. An effective dose from pharmaceutical point of view concerns to a lot of compounds, which is enough to achieve an improvement of the symptoms, for example the treatment, the cure, the prevention or the improvement of certain conditions. The appropriate modalities of administration may include for example the oral administration, rectal and transmucosal or intestinal and the parental administration, including also the administration by intramuscular injections, subcutaneous, intramedullary, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal or intranasal. Subcutaneous or intramuscular administration will be considered to be preferable for patients.
It will be administrated 1-5 ml, preferably 2-4 ml of composition at a distance of 1-4 days. The amount of composition administered may be adjusted by a specialist. This match may be modified during the treatment.
In the case of the traditional methods of treatment is formed both in the case of the primary tumors, as well as in the case of the secondary or of relapse, edemas, causing strong pain when pressing. Against the formation of the edemas is used Cortisone, often even in very high doses. The Cortisones can still cause tumors. Then the Cortisone can create by the action way a strong dependence, so that the remission will be gradual, slow, with partial doses, reduced.
The inventors of the present discovery have ascertained, that the compositions corresponding to the discovery are deranged at their passage into the brain by the Cortisone. The inventors surprisingly found that the extract of incense and/or bamboo can be used as an absolute substitute against the formation of the edemas.
As mentioned above, are taken into consideration especially the preparations of H15 as incense extracts. And the extract of bamboo is in particular the extract from the leaves of bamboo. There can be used directly dry leafs of incense and/or bamboo leaves in the form of capsules, in combination with the pharmaceutical composition, that can be administered according to the present discovery.
In the followings will be presented through examples the production and the effect of the pharmaceutical composition, according to the present discovery. These examples are willing to be exemplary only and will not in any way limit the invention.
For the production of a pharmaceutical composition in the form of an injectable solution, it will be inserted 30 ml into a solution of 0.9% NaCl, 1 ml of saturated solution of peptide toxin HT1 of the Sicarius in the Lachesis D6 DHU (German Homeopathic Union). Finally, it will be added 1 ml of saturated solution of the peptide toxin HT2 of the Sicarius in the Lachesis D6 DHU. It is recommended that both phases to be carried out at the room temperature.
This mixture of cocktail will be completed by the addition of a substance of the Latrodectus geometricus and/or Latrodectus obscurior. In addition to this it can be used the venom of different populations of Latrodectus geometricus and/or Latrodectus obscurior.
Randomly it may be added as immunomodulator 1 ml of Zylexis®, from the company Pfizer®.
Finally, the resulted mixture will be mixed 10 times towards the center of the earth.
The resulting composition can be stored at approximately 70° C., at a place away from light, for 9 months. The loss of the effect, established in experimental way (electrophoretically) after a period of 9 months is of 10%.
In a glass with cell cultures (1250 ml) will be defined the effect of the different extracts of venom through the number of cells after 24, 48 and 100 hours, as it appears in Table 1 below. Here it is visible that the population of cells decreases in an insignificantly number. The decreasement of the population of cells in vitro corresponds to the diminution of a tumor in vivo.
Androctonus
bicoloured
Latrodectus bishopi
Sicarius sp./
Varanus sp.
Latrodectus
Sicarius sp./
curacaviensis
Varanus sp.
Latrodectus dahli
Sicarius sp./
Varanus sp.
Latrodectus erythromelas
Sicarius sp./
Varanus sp.
Latrodectus hasselti
Sicarius sp./
Varanus sp.
Latrodectus katipo
Latrodectus menavodi
Latrodectus rhodesiensis
Latrodectus revivensis
Sicarius sp./
Varanus sp.
Latrodectus schuchi
Sicarius sp./
Varanus sp.
Latrodectus
Sicarius sp./
Varanus sp.
tredecimguttatus
Sicarius sp./
Varanus sp.
Latrodectus variolus
Sicarius sp./
Varanus sp.
Latrodectus hesperus
Sicarius sp./
Varanus sp.
Latrodectus corallinus
Similarly with the comparison Example 1 above have been searched the compositions of the present discovery, as follows in Table 2:
bicoloured
Sicarius oweni
Latrodectus geometricus
Sicarius testaceus
Sicarius hahni
Sicarius albospinosus
Sicarius argentinensis
Sicarius brasiliensis
Sicarius terrosus
Sicarius terrosus formae
Sicarius oweni
Sicarius testaceus
Sicarius argentinensis
Latrodectus obscurior
Sicarius terrosus
Sicarius terrosus formae
Sicarius oweni
Sicarius testaceus
Sicarius argentinensis
Latrodectus geometricus
Sicarius terrosus
Sicarius terrosus formae
Is shown here that after the increased incubation of the cells the growth of the cells does not take place anymore. Usually the toxin from the Sicarius kills in an increased way the tumorous cells. As a passing enzyme is used the phospholipase from the Sicarius argentinensis. This crossing substance is available in sufficient quantity and it enables the diffusion of the peptide toxins, as for example of the Sicarius toxins in the tissues and in the cells.
For the therapy of the brain tumors it will be administrated to the patient every two or three days a subcutaneous injection, of 2-4 ml, of the composition appropriate to the discovery, according to the seriousness of the disease. The development of the therapy will be monitored by tomography with emission of positrons (PET) and the marker of tumors LSA (sialinic acids with lipids). The therapy will match accordingly to the patient's needs.
In addition it will be administrated daily up to 20 tablets of H15 or 3 capsules of incense leaves, in order to prevent the formation of an edema, which can form also in the case of the too accelerate destruction of the tumor.
The capsules containing extracts of incense leaves, are comprised also dry extracts of Bamboo ventricosa.
The conduct of the treatment through various examples of patients:
To separate the factions of effective peptide toxins from the total venom cocktail from the spiders of the Latrodectus and Sicarius species, as well as from the vipers of the Elaphe species, are the preferred procedures of basic chromatographic separation. Of course, the specialist may also apply other procedures of separating the mixture of peptides. The individual fractions, resulting from the separation process are verified in terms of their capacity, or by cell cultures or by experiments with animal. The use of the individual factions of the Latrodectus total cocktail as a crossing agent through the blood barrier of the brain, comes mainly from models of probing on animals.
General representation of a procedure for basic chromatographic separation
For the separation in individual particles/fractions, the total of venom will be inserted into 5 ml of solvent solution for proteins, for the base chromatography, which is composed of 0.25 M Tris/HCL, pH 6.5 up to 7.3, 1.92 M glycine and deionised water. With the total cocktail of venom it will be produced a saturated solution in a recipient of teflon. For the homogeneous mixing of the total cocktail with the solvent solution of proteins, will be shaked for 60 seconds on Vortex, avoiding the foam formation.
After the homogenization, the solution will be casted over an acrylic glass funnel, in a column of transparent acrylic glass, with the interior circumference of 1 cm, the wall thickness of 2 mm and the height of 50 cm, and at the bottom it has a conical shape on the distance of 1.5 mm, but it is open. In the column there are 15 mL Gel (ACA 34; Matrix 3%, Acrylamide 4% Agarose; field of fractioning (MW): proteins from 20-350 kDa, exclusion limit 750 kDa, circumference of the balls: 60-140 μm). The solution of venom casted cranks through the infiltration in the gel, the solution of plugging in the gel. After the total infiltration of the venomous solution in gel will be inserted portioned (without allowing the gel to dry) 165 ml of solvent solution (0.25 M Tris/HCl, pH 6.5-7.3, 1.92 M glycine) in the column. This solvent solution cranks by passage the venomous solution in the gel. The first 15 mL, which will trickle down from the column are the remains of the tampons in the gel and they will be thrown. At the end will be collect 40 fractions of 4 mL.
By the chemical and physical characteristics of this system of separation, results 4 mL for the individual fractions. If in the fractions is really found a single component, this fact will be verified through SDS-PAGE. Only one fascia for each fraction.
For SDS-PAGE will be used as extraction tampon of the fraction in the gel, for the protection of the correlations of peptides and proteins Roti Load 1+2 (Carl Loth GmbH & Co. KG, Karlsruhe: SDS-, Glycerol, blue brophneol, phosphate tampon, Roti Load 1 with mercaptoethanol, Roti Load 2 without mercaptoethanol).
After the separation, the individual fractions will be retained in clean teflon containers, previously sterilized and that can be sealed.
Sicarius spiders are milked, as described in detail above, to extract the total venom cocktail. The venomous total is then extracted by the method of chromatographic separation using the column, for example by HPLC. The fractions 4, 8, 11 and 14 contain substances that destroy the tumorous cells in the subspecies of Sicarius oweni, testaceus, hahni, albospinosus. The fractions 5 and 10 contain substances that destroy the tumorous cells in the South American subspecies of Sicarius argentinensis, brasiliensis, terrosus and terrosus formae.
By the SDS-PAG electrophoresis results by the apparent mass of blue color the following molecular weights (average means) of the frozen-dried substances:
Fraction 4 corresponds to the peptide toxin HT1, and fraction 10 corresponds to the peptide toxin HT2.
Peptide toxins of the Latrodectus and Elaphe were tested as HT1 and HT2 to the venal-glioblastoma standard and cell cultures of astrocytes and also showed the expected effect.
The phospholipase in the total venomous cocktail from the Sicarius argentinensis was extracted and defined in the same way as the peptide toxins. Their molecular weight is 38 000, with which is delimited from other substances from the Sicarius. By the thread of enzyme kinetic checks could be demonstrated the ordering on the type of phospholipase.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2006 060 344.3 | Dec 2006 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2007/064355 | 12/20/2007 | WO | 00 | 5/14/2010 |
