The present invention describes pharmaceutically effective substances from the poison of spiders of the family of Sicariidae as well as their preparation and their use in medicine.

The present invention relates to pharmaceutical compositions containing at least a peptide toxin as well as at least a substance having an antagonistic effect thereon, and/or a penetrant in a pharmaceutically effective amount wherein at least the peptide toxin and optionally the substance having an antagonist effect, and/or the penetrant is derived from the poison of spiders of the family of Sicariidae, as well as to the preparation and the use of the pharmaceutical compositions.

PRIOR ART

In the case of locally manifested tumors, currently, resecting the tumor as completely as possible is the most common form of therapy. Prior to the operation, the tumor is localized using imaging methods and then manually resected by means of an opening intervention. During this, it is impossible to prevent a contact between the operation area and air. It is known from the literature (Stegner H.-E. (1986): Histopathologie der Mammatumoren. Enke Verlag, Stuttgart; Garbe C., Dummer R., Kaufmann R. and Tielgen W. (1997): Dermatologische Onkologie. Springer Verlag, Berlin (see also errata)) that because of the contact with air a metastasis rate of the primary tumor of 93% can be expected.

Other forms of therapy for the treatment of tumors are chemotherapy, irradiation, antibody therapy, cytokine treatment, hyperthermic treatment, or oxygen therapy.

Generally, cell toxins are employed in the chemotherapy of tumors to treat tumors spread throughout the entire body and tumor cells remaining after surgical resection of local tumors (Römpp, Chemielexikon, 9^th edition, vol. 1, 1989, p. 680). Substances used in chemotherapy include for example alkylating substances, anti-metabolites, alkaloids, antibiotics, and hormones (Römpp Lexikon, Biotechnologie und Gentechnik, 2^nd edition, 1999, p. 153). Known as alkylating compounds are for example cisplatin, nitroso urea compounds, or thiotepa. Furthermore, folic acid antagonists, e.g. aminopterine, pyrimidine analogs such as fluorouracil are employed. As antibiotics having an inhibitory effect on DNA-dependent RNA polymerase there may be mentioned bleomycin, doxorubicine, or mitomycin C. Also enzymes such as L-asparaginase have been used in chemotherapy.

The disadvantages of chemotherapy are that it is difficult to use the chemotherapeutics in a site-specific manner and that these cytostatics are extraordinarily severe cell toxins which in addition to the tumor tissue also damage to a great extent healthy tissues including liver and kidney cells. Because of the systemic distribution of the cytostatics it is difficult to judge the side effects arising such as alopecia, vertigo, vomiting, gastro-intestinal bleeding, disturbed circulation etc. (Deutsches Krebsforschungszentrum DKFZ Heidelberg—Focus 19/1995). These numerous, dangerous and undesirable side effects may be explained mainly by an inhibition of the regeneration of quickly proliferating tissues and particularly affect the hematopoietic system, the mucosal and gonadal epithelia, as well as the skin and skin appendages. Among the life-threatening complications infections are the most important, followed by bleeding (Pschyrembel—Klinisches Wörterbuch, 256^th edition, 1990, page 1866).

Irradiation is carried out by means of ionizing radiation wherein generally electron, gamma, neutron, or X-ray beams are used (Zetkin/Schaldach: Lexikon der Medizin, 16^th edition, 1999, page 1922/1923, Ullstein Medical). Similar to chemotherapy, the disadvantage of irradiation is the impossibility to achieve a spatial restriction. Because of the intensity of the radiation also healthy cells and particularly the DNA are severely damaged. Since cancer cells generally divide faster than normal cells, under typical circumstances the cancer cells are the first to be destroyed in radiotherapy. However, there is the risk developing a radiation ulcer (Pschyrembel—Klinisches Wörterbuch 256, edition 1990, page 1602).

Therefore, it is an object of the present invention to provide improved means and methods useful in tumor therapy and/or as an accompanying therapy e.g. in surgical/operative treatment of tumor diseases and which avoid the above-mentioned disadvantages of the prior art.

According to the present invention, this has been achieved by a pharmaceutical composition containing in a pharmaceutically effective amount:

• preparing a raw spider poison mixture by procedures known per se and fractionating the mixture to obtain the peptide toxin and optionally the substance having an antagonistic effect thereon and/or the penetrant and optionally other substances in fractions which are separated from each other if possible;
• combining different fractions of the peptide toxin with fractions containing substances having an antagonistic effect thereon and/or penetrants, or with substances having an antagonistic effect and/or penetrants derived from other organisms to obtain a pharmaceutically effective composition.

The spider poison contains various peptide toxins and various substances having an antagonistic effect thereon and/or penetrants, and optionally other active ingredients which are also relevant in a medical-therapeutical sense. All these substances may be used therapeutically in a pharmaceutical preparation in a specific ratio to be determined by the skilled artisan. While the experiments shown in the Examples are particularly directed to fractions 1 to 12, also the subsequent fractions of the fractionation method specifically described herein contain therapeutically effective substances. It has to be noted that the fractionation procedure merely shows a possibility of how the peptide toxins and the substances having an antagonistic effect thereon may be obtained in an exemplary manner. Also other embodiments are possible.

In this respect, it is preferred that the raw spider poison mixture is prepared from female spiders of the family of Sicariidae. This is advantageous because female spiders of the family of Sicariidae produce a higher amount of poison than male species.

It is further preferred that the raw spider poison mixture is obtained by manual milking. This is advantageous because the raw spider toxin mixture is obtained in a particularly careful manner.

Moreover, it is preferred in the method of the present invention to homogenize the raw spider poison mixture prior to fractionation, and it is further preferred to deep-freeze and further preferred to lyophilize the fractions prior to further processing.

The pharmaceutical compositions of the present invention are suitable for the use in medicine.

According to the present invention, the pharmaceutical compositions may be preferably used in the treatment of tumor diseases wherein a supportive treatment in the case of tumor operations is further preferred.

Specification

At present, about 35,000 species of Araneae exist worldwide. With the exception of approximately 300 species, all of these are actively poisonous animals using their poison for prey catching. Since spiders have only a very small mouth opening, they developed enzymes and poisons to digest their prey outside of the body so that the spiders aspirate the liquefied food. By their poisons, about 50 spider species may also be dangerous to humans. Despite of this, the poisons mainly of these species have been investigated only roughly or not at all. The main components of spider poisons are:
 

• digestive enzymes
• biogenic amines
• organic acids
• peptides
• peptide toxins.

Among the peptide toxins, the following groups of toxins may be found:
 

• heart toxins
• nerve toxins
• blood toxins
• cell toxins
• tissue-destructive poisons.

Initially, the whole poison cocktail of all actively poisonous animals usually achieves a pre-digestion and thereby a specific alteration of the original animal cells by means of an interplay of different substances.

In all spider species used in the present invention in the poison cocktail contains substances which act in a cytotoxic, necrotic and apoptotic manner (digestive action of the poisons). Besides, these also include stopper substances which have an antagonistic effect on these substances having a lytic effect, and/or penetrants.

Since spiders must ingest food which is still useful (whole protein structures and intact amino acids) they have developed their highly effective poison cocktail in the course of their evolution which lasted for 350 millions of years. Within this poison cocktail, the spatial distribution of the peptide toxin is limited depending on the factors of time and concentration by specifically acting enzymes in a controlled manner by an interaction of peptide toxins and substances having an antagonistic effect thereon.

Now, it has been surprisingly found that components of the spider toxins of spiders of the family of Sicariidae may be used for the treatment of tumor diseases.

Because of its lethal effect even in very small doses due to synergisms and antagonisms of various substances contained in the mixture, the poison cocktail as a whole is not useful for pharmaceutical purposes. Secreted by the spiders as a defense poison, one who has been bitten will suffer from the following symptoms:

The bite itself is not recognized by more than 90 percent of the persons bitten. After about 90 minutes a severely suppurating local necrosis of about 3 cm in diameter which already macerates through the skin will appear around the site of the bite. After about 2 hours the bite wound will break open locally, and early systemic effects will be recognized such as circulatory shock and/or heart arrhytmias. After the elapse of further 2-3 hours the lytic substances start their action.

The person bitten will have a strong precipitant urination, the urine being already hemorrhagic. Because poison has an organ necrotic effect, pains will spread throughout the whole abdomen. The liver is unable to metabolize poison in the concentrations and combinations delivered by the spider. If the spider has injected a large amount of poison during biting, the body is unable to manage its degradation and the patient will die because of kidney failure as a result of acute blood poisoning.

However, surprisingly, combinations of peptide toxins and enzymes with opposite action (which have an antagonistic effect on the peptide toxins) and/or penetrants contained in the spider poison wherein at least the peptide toxin and optionally the substance having an antagonistic effect thereon and/or the penetrant is derived from the poison of spiders of the family of Sicariidae may be used in appropriate concentrations and proportions in the treatment of tumor diseases as well as in parallel or in a supportive manner, respectively, in tumor operations, and (residual) tumor tissue may be destroyed. According to the invention, the destruction of tumor tissue which was not resected during operation as well as prevention of the formation of local tumor metastases in the organism may be for example achieved.

Mode of action of the substances used in combination with the peptide toxin:

Antagonistic effect: According to the present invention, tissue may be destroyed without complications in vitro in desired, locally restricted areas, particularly tumor cell predestinated areas. The mechanism of action in this case is based on the native, mutually interacting modes of action of the peptide toxins and of the substances having an antagonistic effect thereon present in the poison cocktail. According to the invention, substances having an antagonistic effect are meant to be substances which are able to digest or to decompose, respectively, the peptide toxin combined therewith. Experiments carried out with human cells revealed the following: a portion of the peptide toxins having a cell destructive effect is distributed in the cell culture faster than the enzymes which digest this toxin and therefore have an antagonistic effect thereon. Based on these findings, a quantitative composition of a combination of peptide toxin and substance having an antagonistic effect may be established depending on the nature and quantity of the tissue area to be lysed in order to achieve a controlled spatial and temporal destruction. By using simple determination of the molecular weight by means of electrophoresis, after contacting in solution the originally employed peptide toxins may no longer be detected in a pre/post contacting comparison (the respective band is missing). In the present invention, the substances having an antagonistic effect are meant to be for example phospholipases and hyaluronidases from spiders of the family of Sicariidae wherein it cannot be ruled out that further substances having an antagonistic effect are present in the spider toxin which may also be used according to the present invention.

Penetration (synergistic effect): The surface protein structure of genetically defective body cells or tumor cells, respectively, is altered (Lottspeich F., Zorbas H. (1998): Bioanalytik. Spektrum Akademischer Verlag Heidelberg Berlin). The phospholipases and/or hyaluronidases employed according to the present invention and optionally additional substances are able to recognize, selectively bind to, and lyse these tumor cells altered in their surface structure. According to the invention this particularly applies to phospholipases. Since the immunogenic state of the phospholipases of animal origin employed alone often is very low as is that of human phospholipases of cancer patients, phospholipases of other organisms, e.g. of spiders or snakes, may be used according to the present invention for the therapy approach for the treatment of tumor diseases. In has been shown by experiments that also non-self phospholipases derived from spider or snake toxins, respectively, not only recognize genetically defective human body cells but also are useful for the infiltration of peptides with necrotic or cytotoxic activities, respectively, to which they are coupled. These peptides, in the present case peptide toxins derived from the poisons of the family of Sicariide, if introduced into the cell, have a cell destructive effect. This effect presumably is an apoptosis because an important marker of apoptosis, caspase-3, may be measured in the medium (supernatant) after cell destruction.

According to the invention, penetrants are meant to be such substances which in combination with the peptide toxin selectively destroy the tumor cells and largely preserve the normal cells. Thus, according to the present invention, this also includes the ability of phospholipases, hyaluronidases and other substances contained in the poison to recognize malignant cells due to their altered surface structure and to dock to these cells and thereby loosen the cell wall, for example for the purpose of actively infiltrating substances which are coupled thereto (preferably peptide toxins having a cell destructive effect) into the malignant cell. In this respect, the penetrants used according to the present invention, particularly phospholipases, act as messengers and adjuvants (synergistic effect). According to the invention, penetration therefore does not refer to a permeability-enhancing effect which in the literature mostly is directed to an enhanced tendency for distribution within tissues.

The peptide toxins having a molecular weight of about 100 kDa have a tissue destructive effect. Because of their high molecular weight and their spatial structure they have a tendency of distribution within tissues of only about 100 cell layers per picogram of substance.

Optionally other substances contained in the raw spider poison mixture may contribute to the effects mentioned.

To avoid undesired cell destruction, an adjustment with respect to the absolute and relative amounts of the components of the peptide toxin/enzyme mixture depending on the type and size of the tumor to be treated may be made according to the present invention in vitro using living human cells (normal and malignant) of the tissue type to be treated. In this respect it is most important to determine the distribution tendency. This may be established in preliminary experiments by comparing the strength of the tumor tissues with that of the tissue surrounding the tumor (see also Example 2).

The mechanism of action of whole animal poison cocktails or of individual substances which have been separated therefrom by column chromatography and characterized via their molecular weight may be performed by testing these in appropriate normal and malignant human cell lines.

According to the present invention, at least the peptide toxin and optionally the substance having an antagonistic effect and/or the penetrant is derived from the poison of spiders of the family of Sicariidae. Preferred are the genera Sicarius, Loxosceles, Scytodes and/or Drymusa. Within the genus Sicarius use of the Sicarius spider species Sicarius oweni, Sicarius testaceus, Sicarius hahni, and Sicarius albospinosus is particularly preferred. Among the spiders of the genus Loxosceles the species Loxosceles rufescens, Loxosceles reclusa, and/or Loxosceles laeta may be used according to the present invention. Among the spiders of the genus Scytodes the species Scytodes thoracica, Scytodes rufa, and/or Scytodes longipes may be used according to the present invention.

According to the present invention, the peptide toxins are preferably derived from the same organism as the substances having an antagonistic effect thereon and/or the penetrants and other active substances which may be optionally contained. In this manner the effective interplay of these substances which has evolved in nature may be utilized.

In another preferred embodiment the substance having an antagonistic effect and/or the penetrant are derived from organisms which are different from spiders of the family of Sicariidae, for example from other spider families, snakes, scorpions etc. In these cases often larger amounts of the substances mentioned may be obtained. Example of such other organisms are cobras, Actrataspis bibronii, Bitis arietans, or Vipera aspis zinnikeri.

The pharmaceutical compositions according to the present invention may be prepared by first preparing a raw spider poison mixture from the spiders using methods known per se and performing a fractionation of the raw spider poison mixture by means of fractionation procedures also known per se for the separation of proteins to obtain the peptide toxins and the substances having an antagonistic effect thereon and/or the penetrants in a form separated from each other as much as possible or in separate fractions, respectively. Subsequently, to prepare a pharmaceutical composition different fractions of the peptide toxin may be combined with fractions containing substances having an antagonistic effect thereon and/or penetrants, or individual fractions of the peptide toxin may be combined with substances having an antagonistic effect and/or penetrants derived from different organisms. Preferably, also snake poisons such as the pit viper snake poison captopril may be contained as the peptide toxins.

Preferably, as the substances having an antagonistic effect also hyaluronidases from snake poisons, for example from cobra poisons, and/or as the penetrants phospholipases from Actrataspis bibronii, Bitis arietans or Vipera aspis zinnikeri, each in combination with one or more fractions of the Sicarius peptide toxin may be used.

For the preparation of pharmaceutical compositions it is also possible according to the invention to combine the fractions additionally with further useful active agents and/or with pharmaceutically conventional carriers and excipients.

For the preparation of the pharmaceutical compositions of the present invention, from the poison cocktail which may be obtained by manual milking of the spider species mentioned above there may be selected, for example via column chromatographic purification, specific poison components (peptide toxins with necrotic and cytotoxic action) as well as natural substances having an antagonistic effect thereon (stopper substances) and/or penetrants of the phospholipase and hyaluronidase type.

The analytics in order to differentiate between the components contained in the fractions may be performed via HPLC-MS-MS (e.g. using an apparatus of Perkin-Elmer company). It has been demonstrated by means of this analysis that based on their backbone structure analyzed by MS-MS the high molecular weight substances are enzymes of the phospholipase and hyaluronidase type. In addition to these enzymes there have also been found polypeptides which must be classified as peptide toxins due to their origin, mode of action and their NX, NHX, NOX, and SX type toxic groups revealed by MS-MS analysis.

The substances used for the pharmaceutical composition according to the present invention may be poisons naturally produced by Araneae of the genus Sicarius which originally have evolved for preying and pre-digestion of animal protein. This natural mode of action may be preserved by a function-preserving, careful preparation of the basic poison substance (e.g. by manual milking).

In contrast to conventional arthropod milking methods by means of an electrical procedure (Weickmann D. (1991): Haltung und Giftigkeit von Sicariidae. Arachnologischer Anzeiger 16:12-13; Weickmann D, Burda R. (1994): Electrophoresis of scorpion venoms. Electrophoresis Forum 1994, Abstracts, Technische Universität München, Oct. 24-26) in which the poison is removed from the animals by an electrical pulse inducing a contraction of the poison glands on the animals (for this purpose, the animals are preferably hypothermic), the poison cocktail is obtained according to the present invention via a manual procedure in which the animals are stimulated to deliver their poison by utilizing a natural defense behavior.

According to an embodiment of the present invention a manual milking method of the spiders is considered. This leads to the preparation of true and pure native poisons in contrast to for example the electrical milking method which due to the electron flow results in restructured substances and molecules, respectively, which may be altered in their action, and wherein substances may be contained in the poisons which the animal normally would not secrete. These substances may, but may not necessarily have a negative effect on the efficiency of the compounds contained in the poison cocktail having a medical effect. An standard analysis and/or quality control of the raw poison mixture may be performed via electrophoretic procedures.
 

Claim 1 of 22 Claims

1. A pharmaceutical composition for the treatment of tumor diseases comprising in a pharmaceutically effective amount:

a) at least one peptide toxin derived from the venom of spiders of the family of Sicariidae having a molecular weight range of about 75 to 175 kDa, as well as

b) at least one enzyme selected from the group consisting of phospholipases and hyaluronidases.

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