MX2007000421A - Carrier for medicaments for obtaining oral bioavailability. - Google Patents

Abstract

The invention relates to a protein complex consisting of at least one hemagglutinin from at least one of the Clostridium botulinum types A, B, C, D, E, F or G and a polypeptide Hc conjugate, whereby the polypeptide Hc conjugate is comprised of a selected polypeptide bound to the heavy chain or the N-terminal fragment of botulinum toxin.

Description

VEHICLE FOR MEDICINES FOR OBTAINING ORAL BIOAVAILABILITY FIELD OF THE INVENTION The present invention relates to a protein complex consisting of at least one hemagglutinin from at least one of the types A, B, C, D, E, F or G of Clostridium botulinum and a conjugate of He of polypeptide, wherein the polypeptide He conjugate consists of a selected polypeptide attached to the heavy chain or its N-terminal fragment of the botulinum toxin. BACKGROUND OF THE INVENTION Although a plurality of pharmaceutical agents is highly active, its therapeutic application is, however, considerably impaired by the fact that these substances can not be administered orally, but only parenterally - what it means by means of injection. In particular, oral administration of protein agents fails because these substances can not reach their site of action via the oral route. After oral administration, the proteins satisfy two obstacles that are difficult to overcome: the denaturing conditions in the gastrointestinal tract lead to an inactivation of the protein, a plurality of proteases promote the degradation of polypeptides, and even if a REF .: 178155 Protein is resistant against these conditions, a substance of high molecular weight is not capable of. overcome the barrier of the intestinal mucosa to pass in the blood and reach the site of action. Proteins are divided by proteases in the stomach and small intestine, respectively, and cleavage products, amino acids and peptides are either absorbed or excreted. Therefore, protein medications have no effect if they are administered orally. Numerous efforts have been made to overcome this disadvantage and make the orally bioavailable protein agents. Some methods are mentioned in this aspect: An attempt has been made to protect the agent from proteolytic degradation by mixtures of protease inhibitors. In this regard, protease inhibitors, such as aprotinin, bestatin, puromycin, soybean trypsin inhibitor, were administered simultaneously with the protein agent; By doing this, the degradation was understood to be prevented and the protein agent was understood to be absorbed without harm. We also tried to modulate the local pH value in the stomach / intestine by the formulations. Even the protein agent itself has been directed to improve the stability of the amino acids by means of their chemical modification, as well as to improve the absorption capacity. The latter was intended to be achieved by the improvement of lipophilicity (for example, by palmitoylation). As an example, the coupling of insulin with an amphiphilic oligomer (hexyl-insulin monoconjugates from NOBEX Corporation) is mentioned. A further method resides in the improvement of mucosal permeability, for example, by the administration of chelators and surfactants, respectively, such as sodium lauryl sulfate, sodium deoxycholate. This concept also comprises the simultaneous administration of concentrated low molecular weight vehicle molecules (e.g., 4- (4-2-hydroxybenzoyl) -aminophenyl) -butanoic acid). An additional method attempts to use specific transport mechanisms in the intestinal wall. In this regard, there is a transport mechanism for the absorption of vitamin B12, which means that it will be used by the protein agents coupling such agents to vitamin B12. Until now, all these different methods have not led to an approved protein medication that is orally bioavailable. An additional method for the delivery of orally bioavailable proteins is described in WO 03/101484. According to the description, the C terminal residue of the heavy chain of botulinum toxin is bound to a polypeptide. The C terminal residue is said to mediate transport through the epithelial membranes. For the oral administration of the hybrid protein, the hybrid protein can be mixed with the auxiliary proteins that naturally surround the botulinum toxin. In addition, WO 02/05844 discloses the supply of orally bioavailable proteins and low molecular weight pharmaceuticals, which are incorporated into a complex of at least one hemagglutinin and one non-haemagglutination protein, potentially non-toxic (NTNH) of the complexes of the Botulinum toxin from Clostridium botulinum. The incorporation of polypeptides that have a molecular weight of <50 kDa, however, has proven that it is not effective enough for profitable marketing. BRIEF DESCRIPTION OF THE INVENTION Therefore, the fundamental problem of the present invention is to provide a means to perform orally bioavailable polypeptides. The problem is solved by the related matter defined in the patent claims. BRIEF DESCRIPTION OF THE FIGURES The following figures illustrate the invention. Figure 1 shows, by means of a diagram, the result of a glucose tolerance test in rats.

Four animals (gavage) were orally treated with 2 U of the He conjugate of insulin and 4 animals with 2 U of the He conjugate of insulin incorporated in the complex (insulin complex). After 60 minutes, the animals were stimulated with 2 g / kg of glucose. Three additional groups were treated with 0.1 U, 0.6 U, 2 U insulin i .p. and they were simultaneously administered also with 2 g / kg of glucose. The glucose level was determined in intervals of approximately 30 minutes. Figure 2 shows, by means of a diagram, the result of a comparison of the effect of the He conjugate of insulin, the He conjugate of insulin integrated in the complex (insulin complex) and the insulin that was applied i .p. The experimental conditions are identical to those of Figure 1. The area under the curve of glucose concentrations (AUC) is shown. DETAILED DESCRIPTION OF THE INVENTION The term "protein complex", as used herein, refers to a vehicle by means of which other selected polypeptides can be transported in the blood system of humans and animals. The protein complex consists of at least one hemagglutinin and one non-haemagglutination protein, potentially non-toxic (NTNH) of the botulinum toxin complexes of at least one of the types A, B, C, D, E, F or G of Clostridium botulinum. The hemagglutinins and NTNH represent the proteins present in Clostridia, whose proteins naturally form the botulinum toxin complex with the botulinum toxin. However, for the sake of clarity, it will be mentioned that the protein complex does not contain a botulinum toxin. The term "botulinum toxin complex", as used herein, refers to a naturally occurring protein aggregate of type A, B, C, D, E, F or G of Clostridium botulinum, which comprises botulinum toxin, hemagglutinin and non-haemagglutination protein, non-toxic (NTNH). The term "polypeptide" or "selected polypeptide", as used herein, refers to a peptide consisting of at least 2 amino acids. The polypeptide can be linear, circular or branched. In addition, the polypeptide can consist of more than one amino acid chain, wherein the chains can be linked together, for example, by a disulfide bond. In addition, the polypeptide may contain modified amino acids and the usual post-translational modifications, such as glycosylation. The polypeptides may be pharmacologically or immunologically active polypeptides or polypeptides used for diagnostic purposes, for example, antibodies. The term "carrier", as used herein, refers to a complete heavy chain (He) or an N-terminal fragment of the heavy chain of botulinum toxin, selected from the botulinum toxin complexes of the types A, B, C, D, E, F or G.

The term "polypeptide He conjugate", "He conjugate" or "conjugate" as used herein, refers to a carrier, which is covalently linked to a selected polypeptide. For example, the insulin He conjugate refers to a molecule consisting of insulin bound to the heavy chain or its N terminal fragment of the botulinum toxin. The term "neocomple or", as used herein, refers to a complex of the protein complex in which a He conjugate is integrated. The bacterium Clostridium botulinum developed an efficient mechanism to pass a protein via the oral route in an organism where the protein is subsequently taken up by its target cells. Such a protein represents the most toxic substance known for now: Clostridium botulinum toxin, also referred to as botulinum toxin hereinafter. In the natural context, botulinum toxin is present in a complex of botulinum toxin with a number of additional proteins expressed by Clostridium um botulinum. If the botulinum toxin complex is administered orally, the botulinum toxin is absorbed from the high molecular weight nerve toxin in the intestine and subsequently reaches the target cell, the motor neuron in the motor endplate. In its site of action, the neurotoxin prevents the release of acetylcholine and, in this way, leads to paralysis of the respective muscle. Clostridium botulinum is divided into 7 serogroups, which are distinguished based on their toxins: Type A, B, C, D, E, F, G. The toxins represent proteins with a molecular weight of approximately 150,000 Dalton (Da). The botulinum toxin complex is usually taken with contaminated food, absorbed enterally and reaches the site of action, the motor endplate. Botulinum toxin consists of two subunits. Each of these subunits satisfies a different function: the heavy chain (molecular weight 100 kDa) binds highly specifically to the nerve cell and subsequently allows the translocation of the light chain in the cytoplasm of the cell. In the native botulinum toxin, the heavy chain (He) is bound to the light chain (Le) by a disulfide bridge. The light chain serves as a protease, which divides the proteins (SNARE proteins) that are responsible for the fusion of the secretory vesicles with the membrane of the nerve cell. Therefore, the secretory vesicles are not able to release acetylcholine: muscle activation is blocked. Both chains originate from the polypeptide originally synthesized by the proteolytic cleavage. In the case of some types of Clostridia, the division is already carried out by the proteases characteristic of the Clostridia (type A, C, partially B), while in the case of other types the division is carried out in the tract intestinal (trypsin) or only in the tissue of the recipient. The heavy chain in the isolated form and without any contamination by the light chain or by the complete toxin is absolutely non-toxic; by itself, it is not able to block the release of acetylcholine in nerve cells. Clostridia synthesize a number of additional proteins that form a complex with the botulinum toxin (botulinum toxin complex), which is stable in an acidic environment and protects the neurotoxin from denaturation and proteolytic degradation and, in addition, allows the uptake through the intestinal mucosa. The additional proteins, referred to herein as "complex proteins" hereinafter, represent a number of hemagglutinins and a non-haemagglutination, non-toxic protein (NTNH) having a molecular weight of about 120,000 Da. The additional proteins form the protein complex. In the case of the botulinum toxin type A complex, the following haemagglutinins were described: Ha2 having approximately 16,900 Da, Ha3a having approximately 21,000 Da, Ha3b having approximately 52,000 Da and Hal having approximately 35,000 Da. Botulinum toxin complexes of types B to G are synthesized following a similar scheme. As an example, the B botulinum toxin complex may be mentioned. In this case, they are described separately from NTNH, HA-70 having a molecular weight of about 70,000 Da, Ha-17 having a molecular weight of about 17,000 Da and Ha-33 having a molecular weight of approximately 33,000 Da (see Bhandari, M. et al. (1997) Currenü Microbiology 35, pp. 207-214). In addition, East, A. K.. et al., (1994) Sys tem Appl. Microbiol. 17, pp. 306-312) describe the sequence of Ha-33 of type B compared to the sequence of type A and C. For type C and type D are also described, in addition to Ha 33 (= Hal), - analogously to type A - a Ha3b having a molecular weight of approximately 33,000 Da, also Ha3b having approximately 53,000 Da and Ha3a having approximately 22-24,000 Da and Ha2 having approximately 17,000 Da (cf. Inoue, K. et al., (1999) Microbiology 145, pp. 2533-2542). Surprisingly, the inventors have found in the reconstitution experiments that only the heavy chain of botulinum toxin, which means without the light chain, alone or coupled to a polypeptide, is quantitatively incorporated into the protein complex. The polypeptide can be chemically coupled to the heavy chain of a botulinum toxin and integrated into a protein complex consisting of at least one complex protein. A plurality of chemical methods are available for coupling the protein to the heavy chain. There is a broad spectrum of bifunctional reagents that allow the union of two different proteins. Preferably, reagents that establish a disulfide bridge with a cysteine of the coupling partner are used. Subsequently, the binding to the vehicle, the heavy chain, can be carried out in the next step. Suitable reagents for such coupling are, for example, reagents, such as SPDP (N-succinimidi-3- [2-pyridyldithio] -propionate) or DTDP (4,4'-dithiodipyridine), in case only one disulfide bridge without a spacer between proteins. Such coupling has the advantage that it can be divided in vivo under reducing conditions, for example, in the cytoplasm by means of the thioredoxin system. In this case, the coupling is selected such that the incorporation of the heavy chain into the protein complex does not interfere with, and the biological activity of the polypeptide is maintained. Alternatively, coupling of the heavy chain with the polypeptide can be obtained by synthesizing both peptides as a recombinant fusion protein in an appropriate expression system. The polypeptides attached to the carrier are preferably pharmacologically or immunologically active polypeptides, which are administered orally by means of the protein complex according to the present invention, which may be therapeutically or prophylactically active. The polypeptides selected may be, for example, hormones, cytosines, enzymes, growth factors, antigens, antibodies, inhibitors, receptor agonists or antagonists or coagulation factors. In this regard, it is not decisive whether the polypeptides have been produced or isolated recombinantly from their natural sources. Preferred polypeptides are insulin, erythropoietin, interferons, interleukins, HIV protease inhibitors, GM-CSF (granulocyte-macrophage stimulation factor), NGF (nerve growth factor), PDGF (platelet-derived growth factor), FGF (fibroblast growth factor), plasminogen activators, for example, TPA (tissue plasminogen activator), renin inhibitors, human growth factor, IGF (insulin-like growth factor), vaccines, such as tetanus vaccine , hepatitis B vaccine, diphtheria vaccine, antibodies, for example, herceptin (antibody against Her2), antibodies against TNF (tumor necrosis factor), antibodies against the EGF receptor, antibodies against VEGF, antibodies against IgE, antibodies against CDlla, calcitonin, urokinase, streptokinase, angiogenesis inhibitors, factor VIII, factor Xa antagonists, metalloprotease inhibitors.

Polypeptides used for diagnostic purposes can be, for example, antibodies or ligands, wherein the polypeptides can be provided with a tag. As a brand, any brand that can be detected in the body of a human or animal can be considered. The preferred brands are isotopes, for example, C13 or radioactive labels. The labeled antibodies can be used for the detection of tumors; the labeled ligands can be used for the detection of, for example, pathological receptors. The vehicle bound to the polypeptides is incorporated into the protein complex. The protein complex is composed of at least one hemagglutinin and, if desired, at least one NTNH. In this regard, hemagglutinins and NTNH are selected from botulinum toxin complexes that occur naturally of types A, B, C, D, E, F or G of Clostridium um botulinum. However, the protein complex may contain a composition that differs from its natural composition, for example, it may be composed solely of hemagglutinin without the NTNH proteins. In addition, the protein complex may be composed of fewer naturally occurring hemagglutinin species than the botulinum toxin complex, preferably of three different haemagglutinin species, preferably two, in particular a haemagglutinin species is preferred, where in each case the protein complex may contain the NTNH protein or not. In addition, the protein complex may be composed of a mixture of one or more hemagglutinin species and / or NTNH proteins of different serotypes. Protein complexes are preferred which correspond to naturally occurring protein complexes (without botulinum toxin) of Clostridium botulinum of types A, B, C, D, E, F or G, for example, a protein complex with Hal , Ha2, Ha3a, Ha3b and NTNH of Clostridium botulinum type B. In addition, the protein complex can be composed of Hal, Ha2, Ha3a and NTNH, of Hal, Ha2, Ha3b and NTNH, as well as of Hal and Ha3a, Ha3b and NTNH, in addition to Ha2, Ha3a, Ha3b and NTNH, of Hal, Ha2 and. NTNH, of Hal, Ha3a and NTNH, of Hal, Ha3b and NTNH, of Ha2, Ha3a and NTNH, of Ha2, Ha3b and NTNH, of Ha3a, Ha3b and NTNH or of additional arbitrary combinations of the complex proteins listed. In addition, the protein complex may be composed of one of the hemagglutinins and NTNH; in addition, the protein complex may be composed of the listed combinations of hemagglutinins without NTNH. In accordance with exemplary protein complexes of type B, protein complexes of haemagglutinins and / or NTNH of types A, C, D, E, F or G are also preferred. A further aspect of the present invention relates to providing a method for the production of the protein complex according to the present invention, the method comprises the following steps: a) isolation separated from at least one botulinum toxin complex of type A, B, C, D, E , F or G of Clostridium botulinum at a pH value in the range of 2.0 to about 6.5, b) increasing the pH value to a value in the range of about 7.0 to about 10.0, c) removing the respective botulinum toxin from complex proteins by means of chromatographic methods, d) mixing the complex proteins obtained in step c) with a selected polypeptide He conjugate or e) separating the complex proteins obtained in step c) and mixing p or at least one complex protein with a polypeptide He conjugate; f) dialyzing the mixture of step d) or e) against a buffer at a pH value in the range of about 6.5 to about 2.0, preferably over a range of about 4.0 to about 6.0, particularly preferred to 6.0. Complex proteins can be isolated from natural botulinum toxin complexes. An example of a method for isolation is as follows: First, the botulinum toxin complex is isolated from clostridia at an acidic pH value, preferably at a value in the range of about 2.0 to about 6.5, is preferred in particularly in the range of about 4.0 to about 6.5, particularly preferred at a pH of 6.0. After increasing the pH value to a value in the range of about 7.0 to about 10.0, preferably at a pH value in the range of about 7.0 to about 8.0, the botulinum toxin is removed by the chromatographic methods, which they are common in protein chemistry. Such a method can be worked because the complex is stable at a pH value <6.5, disintegrates at a neutral and alkaline pH value, respectively and the toxin is released. The toxin-free complex proteins can then be mixed with a polypeptide He conjugate, and the pH value can be reduced by dialysis against a buffer, which is common in protein chemistry, particularly preferred against a phosphate buffer, acetate or citrate, at a pH value in the range of about 2.0 to about 6.5, preferably in the range of about 4.0 to about 6.0, is particularly preferred at a pH of 6.0. In this manner, a protein complex containing the polypeptide He conjugate is formed and thereby provides the oral bioavailability of the selected polypeptide. Other chromatographic methods, concentration methods and precipitation, which are common in protein chemistry, can also be used for the isolation of complex proteins. Due to their known DNA sequences, complex proteins can also be produced recombinantly by DNA recombination techniques, especially host organisms. The complex proteins produced in such a way can also exhibit modifications, which means that they can be derived from the complex proteins. In this aspect, the modifications do not only mean eliminations, additions, insertions or substitutions, but also also the chemical modifications of the amino acids, for example, methylations or acetylations, as well as post-translation modifications, for example, glycosylations or phosphorylations. The expression of the desired proteins in different hosts is well known to those skilled in the art and need not be described separately herein. In this regard, the complex proteins necessary for the protein complex can be expressed separately or simultaneously in a host organism. The production of recombinant complex proteins in bacteria, for example in E. coli, Bacillus subtilis or Clostridium difficile, or in eukaryotic cells, for example, in CHO cells, in insect cells, for example, using the Baculovirus system, is preferred. or in yeast cells. The complex proteins can be isolated and mixed with the polypeptide conjugate selected according to the method described above. In addition, the selected polypeptide He conjugate can be simultaneously expressed as a fusion protein together with the complex proteins in the host organism. In particular, the simultaneous or separate production of the respective complex proteins together with the polypeptide He conjugate selected by means of a YAC in yeast is preferred. In addition, the protein complexes according to the present invention may be composed of a mixture of recombinantly produced complex proteins and complex proteins isolated from botulinum toxin complexes. The following examples illustrate the invention and are not constructed to be limiting. Example 1: Isolation of heavy chain of Clostridium botulinum type A toxin Clostridium botulinum type A toxin (strain ATCC 3502) was cultured according to (published methods exhibiting the modifications as listed as follows (cf. Das Gupta &Sathyamoorthy, 1984, Toxicon 22, pp. 415-424) After 72 hours of growth, the toxin was precipitated by the addition of 3N sulfuric acid After the extraction of the precipitate and the removal of the acids The toxin was precipitated by means of ammonium sulfate After stabilization and dialysis, a DEAE-sepharose chromatography (2.6 x 15 cm) was performed at pH 6.0, the bound toxin was eluted with 150 mM NaCl, dialysed against 50 mM Tris / HCl and subjected to an additional ion exchange chromatography using a sepharose Q column (2.6 x 10.0 cm) The neurotoxin was eluted with a gradient of NaCl (0-300 mM NaCl). the neurotox ina met and dialyzed against 10 mM Na phosphate, pH 7.0. The dialyzate was applied to a sepharose S column (1.6 x 11 cm) and eluted with a gradient of NaCl (0-300 mM NaCl). Chromatography produced the high purity neurotoxin. The isolation of the heavy chain of the neurotoxin was carried out through published literature that exhibits the modifications as listed below (see Kozaki et al., 1981, J. Med. Sci. Biol. 34, pp. 61- 68). In this regard, the high purity neurotoxin type A was dialyzed against a borate / phosphate buffer, pH 8.5 and bound to a column (1 x 5 cm) filled with QAE-Sephadex. After washing with the borate / phosphate buffer, which also contained 10 mM DTE, the column was incubated with 3 mL of the borate / phosphate buffer overnight, which also contained 150 mM DTE and 2 M urea. After elution Subsequent light chain with the borate / phosphate buffer + 10 mM DTE + 2 N urea, the heavy chain mixed with the same buffer was eluted with 200 mM NaCl.

To remove the incompletely removed active neurotoxin, the pooled fractions were pumped four times on an affinity column. The affinity column was filled with sepharose, to which an antibody against Clostridium botulinum type A light chain was coupled. After chromatography, a heavy chain was available, which did not contain contamination with light chain or toxin. native Even at high concentrations, no activity was shown in the activity test (mouse diaphragm test). Example 2: Preparation of complex Clostridium um botulinum type B proteins Clostridium botulinum type B complex proteins are isolated after fermentation of Clostridium botulinum type B (strain Okra) according to a published method (cf. et al., 1986, European Journal of Bioch. 154, pp. 409-416), where some steps were modified: As in Example 1, the nucleic acids were precipitated from the extract after the extraction of the precipitated acid biomass, and the complex of the toxic botulinum toxin was precipitated from the supernatant with ammonium sulfate. The precipitate was resuspended in 0.05 M sodium citrate + 1 mM EDTA, pH 5.5 and purified using DEAE-sephadex chromatography after dialysis, where the high molecular weight complex did not bind to the column (5 x 12 cm ) and passed quantitatively through the column. The eluate containing the complex was subjected to chromatography using a sepharose Q column after dialysis against 50 mM Tris / HCl, 1 mM EDTA, pH 7.9, where the complex proteins did not bind to the column, whereas the neurotoxin remained attached to the column and eluted only with a saline gradient. Further purification of the complex proteins was performed on a Q Hyper D column (2.6 x 13 cm), which was equilibrated with the same buffer (50 mM Tris / HCl, 1 mM EDTA). The toxin-free protein complex was eluted with a gradient of sodium chloride (0-300 mM NaCl). The last traces of neurotoxin were removed by means of affinity chromatography. For this purpose, the solution containing the complex was pumped four times onto a column which contained an affinity sepharose matrix coupled to an antibody (IgG fraction) against the botulinum neurotoxin type B. Subsequently, biological activity was no longer detectable ( toxicity) in the activity test (mouse hemi-diaphragm test). Example 3: Integration of the heavy chain of botulinum toxin type A into a protein complex of C. botulinum type B 100 μg (34 μl) of the highly purified complex proteins type B of the example, was mixed with 200 μg (690 μl) of the isolated heavy chain type A of example 1. The mixture was dialyzed for 3 days at 2-8 ° C against 1150 mM Na phosphate buffer, pH 6.0 with 150 mM NaCl. Then, 450 μl was precipitated by the addition of 150 μl of 4M ammonium sulfate. Under these precipitation conditions, the heavy chain, which is not incorporated in the protein complex, remains in solution, while the protein complex (with or without the incorporated chain) precipitates quantitatively. After re-incubation overnight, the granulate was centrifuged and resuspended in 120 μl of Na phosphate, NaCl mm, 2 mM EDTA, pH 6.0. Protein complex formation was tested by "gel filtration" through BioSep S-3000. The chromatogram showed only a single peak (at a molecular weight of approximately 500,000 Daltons). SDS-polyacrylamide gel electrophoresis showed that the peak fractions contained the complex proteins of type B and the heavy chain of botulinum toxin type A. Example 4: Coupling of insulin to the botulinum toxin heavy chain of type A. Synthesis of SPDP-Insulin 12.5 mg of insulin (Roche, recombinant) were dissolved in 6.3 ml of buffer (10 mM sodium carbonate), pH = 6.9). For this purpose, 3 μl of the citaconanhydride (Fluka) was taken with a pipette to block the amino groups and the mixture was incubated at room temperature, where the pH value was observed. By adding 1 M NaOH, the pH value was maintained at 6.8-7.0. The reaction mixture was subsequently dialyzed overnight at 4 ° C against 50 mM sodium phosphate, 100 mM sodium chloride, pH = 7.3. 6.2 mg of SPDP (N-succinimidyl 3- [2-pyridyldithio] -propionate, Pierce) were dissolved in 500 μl of DMF. 274 μl of this solution was added to 6.3 ml of insulin derivative (adjusted to pH 8.3) and the mixture was incubated at room temperature in a mixer for 1 hr. The remaining SDPS was removed by dialysis against 50 mM sodium phosphate, 100 M NaCl, pH = 7.3. To remove the protective groups, dialysis against water (2 h at room temperature) and then against 10 mM HCl at room temperature for 5 y? h. Finally, dialysis was performed against 50 mM sodium phosphate, 100 mM sodium chloride, 4 mM EDTA, pH = 7.3 overnight (final volume of the SPDP solution: 7 ml). Production of the insulin He conjugate 25 mg of the botulinum toxin type A heavy chain, which was isolated according to example 1, was mixed with 8 mg of SPDP insulin (final volume: 41.5 mg) and incubated at 4 ° C for 4 days (extreme mixer on end). For the removal of the uncoupled insulin-SPDP, dialysis was performed against 50 mM Tris / HCl, 250 mM NaCl, 1 mM EDTA, where the dialysis hose exhibited an exclusion limit of 50 kD. The product was analyzed by Western Blot. Using antibodies against insulin, a protein having the molecular weight of ~ 100 kD was identified. From here, the conjugate of the heavy chain and insulin (= He conjugate of insulin) was represented. The activity of insulin in vi tro was detected using 3T3 cells. Example 5: Incorporation of the He conjugate of insulin into the Clostridium botulinum type B protein complex 7.4 mg of the insulin He conjugate (from Example 4) were mixed with 21 mg of the protein complex (purified according to example 2) in 52 ml and dialyzed against 50 mM sodium phosphate, 250 mM NaCl, 1 mM EDTA, pH 6.0 for 4 days at 4 ° C. A 500 μl sample was dialyzed by gel filtration using a BioSep S-3000 column (Phenomenex). Only a high molecular weight peak can be shown at a molecular weight of -600 kD. A sample of the peak fraction was analyzed by Western Blot. Using antibodies against insulin, it was shown that insulin (as insulin conjugate) was integrated into the protein complex (consisting of the complex proteins of C. jbotulintim).

Example 6: Testing an insulin neocomplex in rats using the glucose tolerance test The effectiveness of a He conjugate of insulin incorporated in a protein complex (hereinafter referred to as the insulin neofycomplex) after oral administration is tested in an animal experiment in rats. Groups of 4 rats were treated with the insulin neocomplex or the insulin free He conjugate (without the complex proteins). Untreated animals served as control. The insulin dose was the same in both groups and was 2 U / animal. The solutions were administered by means of a gavage. As additional controls were administered 0.1 U, 0.6 U and 2.0 U of insulin i. p. 1 h after the application of the insulin neo-complex and the insulin-free He conjugate the animals were stimulated with glucose (2 g / kg orally). In this interval, the positive control injection was also carried out (0.1 U, 0.6 U and 2.0 units of insulin). After 30, 60, 120 and 180 minutes the blood was taken and the glucose level was determined. After 60 minutes a maximum glucose concentration was reached with approximately 140 mg / ml in the case of the control animals, whereas in the case of the animals treated with the insulin neo-complex, the concentration increased only slightly (see Figure 1). If the progress of the glucose concentration (area under the curve, AUC) is considered, the effect of the insulin neocomplex is comparable with the administered insulin i. p. (Figure 2). The concentration of glucose in the case of the animals that were treated with the He conjugate of insulin behaved only as in the case of the control: the concentration increased strongly up to the interval of 60 minutes. From this, it can be concluded that the He conjugate of insulin only has no effect on the insulin level; therefore, administration of the insulin He conjugate without incorporation into the protein is not appropriate to make the insulin orally bioavailable. Example 7: Comparison of the integration of the He conjugate of insulin with the insulin bound to the C-terminus of the heavy chain of the botulinum toxin The C-terminus fragment of the heavy chain (Mr «50 kD, also referred to hereinafter as fragment Hl) of botulinum toxin type A, was expressed recombinantly in E. coli. For this purpose the DNA sequence of the C terminal fragment of botulinum toxin type A 871-1296 aa (NIINT ERPL) bound to a His tag was cloned into vector pBN29 4772; E. coli Ni5 [pREP4] (Quiagen) was transformed therewith and the expressed fragment was purified by affinity chromatography using a Ni-NTA-Sepharose column. 100 μg of the toxin free complex proteins of C. botulinum type B were mixed with 200 μg (340 μl) of the recombinant C terminal fragment and dialyzed at 2-8 ° C against 50 mM Na phosphate, pH 6.0 with NaCl 150 mM for 3 days. Then, the reaction was brought to 450 μl with water and precipitated by the addition of 150 μl of 4M ammonium sulfate. Under these precipitation conditions, the C-terminal fragment, which is not incorporated into the protein complex, remains in solution, while the protein complex (with or without the incorporated C-terminal fragment) precipitates quantitatively. After overnight re-incubation, the pellet was centrifuged and resuspended in 150 μl of 150 mM Na phosphate, 150 mM NaCl, 2 mM EDTA, pH 6.0. 100 μ were separated on a BioSep S-3000 gel filtration column in the complex and the unbound C-terminal fragment, which is potentially present as contamination. Two peaks were eluted: the first peak (12.2 minutes) represents the protein complex. A very small peak represents the free C-terminal fragment fragment. The first peak was examined using SDS-PAGE; only complex proteins were present; therefore, the C-terminal fragment was not integrated into the complex but remained unbound in solution during the precipitation of ammonium sulfate. In conclusion, the C terminal fragment of botulinum toxin can not be integrated into the complex.

Example 8: Comparison of the biological activity of insulin conjugate Hl and He conjugate of insulin The C terminal fragment (fragment Hl) of the heavy chain of botulinum toxin type A was produced as described in example 7. After of the derivation, 13 mg of the Hl fragment was mixed with 8 mg of insulin-SPDP. The insulin-SPDP production was carried out analogously to example 4. After incubation for 24 hours at 4 ° C, the uncoupled insulin-SPDP was removed by dialysis against 50 mM Tris / HCl, 250 mM NaCl, EDTA 1 mM. 3.8 mg (insulin conjugate Hl) were dialyzed with 21 mg of the toxin-free protein complex (purified according to example 2) against 50 mM Na phosphate, 250 mM NaCl, 1 mM EDTA, pH 6.0 for 5 days at 4 ° C as in the case of the integration of the heavy chain in the complex (example 5). This mixture was tested using animal experiments. Groups of 4 rats were treated with the He conjugate of insulin (example 5) or with the Hl conjugate of insulin + complex proteins or with saline. The dose of the first two groups was 2 U of insulin per animal. The stimulation with glucose (0.5 g / kg i .p.) Was carried out 1 h after the administration. After 30 minutes the measurement of the sugar level showed 170 ± 12 mg / dl in the case of the animals that received salt, while the increase in the group, which received the HC conjugate neocomplex, only increased to 115 ± 14 mg / dl. The initial value in the glucose injection interval was 85 ± 14 mg / dl. In conclusion, the Hl conjugate of insulin with the complex proteins showed no effect in the animal experiment. Example 9: Use of an insulin conjugate with the C-terminal shortened heavy chain To obtain a chain, which is C-terminally shortened by 30 amino acids, the chromosomal DNA was prepared from a culture of C. botulinum type A (ATCC 3502). By PCR amplification, a fragment of the gene encoding the light chain, this fragment further contains a thrombin cleavage site in the region of the loop, was cloned into the plasmid pQE60 (pQE-BoNT (A) -L). A gene fragment was generated from the chromosomal DNA by PCR amplification encoding a chain, which is C-terminally shortened by 30 amino acids. This gene fragment was cloned to the 3 'end of the gene fragment encoding BoNT (A) -L in the expression plasmid pQE-BoNT (A) -L (pQE-BoN (A) -L H30min). The expression strain of E. coli M15 [pREP4] (Qiagen) was transformed by this plasmid. After induction with 500 μM IPTG (25 ° C overnight) the cells were lysed and subjected to chromatography using a Ni-NTA-agarose column. Of the toxin that was obtained in such a manner and which is shortened by 30 amino acids, the heavy chain H30min was isolated as described in example 1. The heavy chain H30min was conjugated with insulin in a manner analogous to example 4 and integrated into the toxin-free complex of C. botulinum type B in a manner analogous to example 5. The neocomplex synthesized in such a way was tested using the glucose tolerance test with 4 animals as compared to a control without the complex (according to the example 8). At a dose corresponding to 2 U of insulin, the blood sugar level increased to 110 ± 18 mg / dl after 30 minutes, while it increased to 168 ± 14 mg / dl in the case of the control animals ( initial value 90 ± 5 mg / dl).

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (12)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A protein complex consisting of at least one hemagglutinin of at least one of the types A, B, C, D, E, F or G of Clostridium botulinum and a polypeptide He conjugate, characterized in that the polypeptide He conjugate consists of a selected polypeptide linked to the heavy chain or its N terminal fragment of botulinum toxin.
2. 2. The protein complex according to claim 1, characterized in that the haemagglutinins are a mixture of hemagglutinins of at least one of the types A, B, C, D, E, F or G of Clostridium um botulinum.
3. 3. The protein complex according to any of the preceding claims, characterized in that the protein complex also contains a non-toxic, non-haemagglutination protein.
4. 4. The protein complex according to any of the preceding claims, characterized in that the heavy chain is isolated from one of the botulinum toxin complexes of types A, B ,. C, D, E, F or G. The protein complex according to any of the preceding claims, characterized in that the heavy chain is produced recombinantly in an appropriate expression system. 6. The protein complex according to any of the preceding claims, characterized in that the polypeptide is a hormone, a cytosine, a growth factor, an antigen, an antibody, an inhibitor, a receptor agonist or antagonist or a coagulation factor . The protein complex according to any of the preceding claims, characterized in that the selected polypeptide and the heavy chain or its N-terminal fragment are produced recombinantly as a fusion protein. The protein complex according to any of claims 1 to 6, characterized in that the selected polypeptide and the heavy chain or its N-terminal fragment are linked by a chemical bond. 9. The protein complex according to claim 8, characterized in that the chemical bond is a disulfide bond. 10. The protein complex according to claim 8, characterized in that the chemical bond is a peptide bond. 11. Method for the production of a protein complex according to any of the preceding claims, characterized in that it comprises the steps of: a) isolating a botulinum complex of Clostridium um botulinum at a pH value in the range of 2.0 to 6.5; b) adjusting the botulinum complex isolated from step (a) to a pH value in the range of 7.0 to 10.0; c) remove botulinum toxin from complex proteins by means of chromatographic methods; d) mixing the complex proteins obtained in step c) with a polypeptide He conjugate; or e) separating the complex proteins obtained in step c) and mixing at least one complex protein with a polypeptide He conjugate; f) dialyzing the mixture of step d) or e) against a buffer at a pH value in the range of 6.5 to 2.0. 12. Use of the protein complex according to any of claims 1 to 10 as a transport vehicle for the pharmacologically active, immunologically active polypeptides or for the polypeptides used for diagnostic purposes.