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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/04—Centrally acting analgesics, e.g. opioids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/06—Antimigraine agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/24—Antidepressants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/26—Psychostimulants, e.g. nicotine, cocaine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/24—Metalloendopeptidases (3.4.24)
  • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to a transport protein that binds to higher or lower affinity neurons than the neurotoxin produced by Clostridium botulinum.
• the transport protein is preferably taken up by receptor-mediated endocytosis.
• This protein finds utility as a transporter that translocates other chemical substances (e.g., proteases) from the acidic endosomal compartment into the cytosol of neurons that can not penetrate physiologically through the plasma membrane into the cytosol of nerve cells.
• the present invention relates to the use of a transport protein for the introduction of inhibitors of neurotransmitter secretion.
• Exocytosis refers to the fusion of the membranes of intracellular vesicles with the plasma membrane. In this process, the vesicular content is simultaneously released into the synaptic cleft. The fusion of the two membranes is regulated by calcium, which reacts with the protein synaptotagmin. Together with other cofactors, synaptotagmin controls the status of three so-called fusion proteins, SNAP-25, synaptobrevin 2 and syntaxin IA. While syntaxin IA and synaptobrevin 2 are integrated into the plasma and vesicle membranes, SNAP-25 is only weakly bound to the plasma membrane.
• the above-mentioned fusion proteins are the target molecules (substrates) of the light chain (LC) of the clostridial neurotoxins formed by the bacteria C. botulinum, C. butyricum, C. baratii and C. tetani.
• the anaerobic, gram-positive bacterium C. botulinum produces seven different serotypes of clostridial neurotoxins. These are referred to as the botulinum neurotoxins (BoNT / A to BoNT / G). Of these, BoNT / A and BoNT / B in particular cause a neuroparalytic disorder in humans and animals called botulism.
• the spores of C. botulinum are found in the soil, but may develop into improperly sterilized and sealed home-grown canned foods, which are responsible for many of the botulism cases.
• BoNT / A is the most active of all known biological substances. Only about 5-6 pg of purified BoNT / A represents an MLD (median lethal dose).
• a Unit (U) of BoNT / A is defined as the MLD, which after intraperitoneal injection kills half of female Swiss Webster mice weighing 18-20 g each. Seven immunologically different BoNTs were characterized. They bear the names BoNT / A, B, Cl, D, E, F and G and can be distinguished by neutralization with serotype-specific antibodies. The different serotypes of BoNT differ in affected species with regard to the severity and duration of the caused paralysis. So z. B. in the rat with regard to the paralysis BoNT / A 500 times more effective than BoNT / B.
• BoNT / B has been found to be non-toxic in primates at a dose of 480 U / kg body weight. The same amount of BoNT / A is equivalent to 12 times the lethal dose of this substance in primates. On the other hand, in mice the paralysis time after injection of BoNT / A is 10 times longer than after injection of BoNT / E.
• BoNTs are used to treat neuromuscular disorders caused by hyperactivity in skeletal muscle caused by pathologically overactive peripheral nerves are characterized.
• BoNT / A is approved by the US Food and Drug Administration for the treatment of blepharospasm, strabismus, hyperhidrosis, wrinkles and hemifacial spasm.
• the remaining BoNT serotypes apparently have a lower potency and shorter duration of action.
• Clinical effects of peripheral intramuscularly administered BoNT / A usually occur within one week.
• the duration of symptom suppression by a single intramuscular injection of BoNT / A is usually about three to six months.
• BoNT / A, Cl and E cleave SNAP-25, while BoNT / B, D, F, G and tetanus neurotoxin (TeNT) attack the vesicle-associated membrane protein (VAMP) 2 - also called synaptobrevin 2.
• VAMP vesicle-associated membrane protein
• BoNT / Cl also splits syntaxin IA.
• Clostridia bacteria release the neurotoxins as single-chain polypeptides, each with 1251 to 1315 amino acids. Subsequently, endogenous proteases of each of these proteins cleave at a given site in two chains ('nicking'), but the two chains remain connected by a disulfide bridge. These two-chain proteins are referred to as holotoxins (see Shone et al., (1985) Eur. J. Biochem., 151, 75-82). The two chains have different functions. While the smaller segment, the light chain (LC), represents a Zn 2+ -dependent endoprotease, the heavy chain (HC) is the light chain transporter.
• LC light chain
• HC heavy chain transporter
• the HC binds with the Hc fragment to neuronal gangliosides, is taken up by the receptor-mediated endocytosis into the cell interior and reaches the natural vesicle circulation via the endosomal compartment.
• the H N fragment invades the vesicle membrane and forms a pore. Any substance (X) that is linked to the HC through a disulfide bridge will be separated from the HC by intracellular redox systems that gain access to and reduce the disulfide bond. Ultimately, X will appear in the cytosol.
• the HC is the carrier of an LC, which in the final step cleaves its specific substrate in the cytosol.
• the cycle of complex formation and dissociation of the fusion proteins is interrupted and thus inhibited the release of acetylcholine.
• striped muscles are paralyzed, and sweat glands cease their secretion.
• the duration of action of the individual BoNT serotypes varies and depends on the presence of intact LC in the cytosol.
• the preferential blocking of cholinergic transmission can be explained by the HC entering the neuron at the periphery. Central synapses are protected by the blood-brain barrier, which proteins can not overcome.
• BoNT / A complex also called progenitor toxin A, has been used in the recent past to treat motor dystonia, as well as to attenuate excessive sympathetic activity (see Benecke et al., 1995, Act Neurol., 22, 209ff) and for alleviation pain and migraine (see Sycha et al., 2004, J. Neurol., 251, 19-30).
• This complex consists of the neurotoxin, various hemagglutinins, and a non-toxic, non-hemagglutinating protein. Under physiological pH, the complex dissociates in a few minutes. The resulting neurotoxin is the only component of the complex that is therapeutically relevant and causes symptom relief. Since the underlying neurological disease is not cured, the complex must be re-injected at intervals of three to four months. Depending on the amount of injected foreign protein, some patients will form specific BoNT / A antibodies. These patients become resistant to the neurotoxin. Once antigen-sensitive cells have recognized the neurotoxin and antibodies have been generated, the memory cells in question will remain for years. Therefore, it is important to treat the patients with drugs of highest activity in the lowest possible dosage.
• the preparations should also contain no further proteins of bacterial origin, since they can act as immune adjuvants. Such substances attract macrophages that recognize both the immune adjuvants and the neurotoxins and present to the lymphocytes, which then respond to the formation of immunoglobulins. Consequently, only products of the highest purity, which do not contain foreign proteins, should be used for therapy.
• the resistance of the patients to the neurotoxin is based, on a molecular level, predominantly on the presence of neutralizing antibodies.
• a transport protein (Trapo) is now presented, which can overcome the above-described problems of the previously known methods.
• This object has been achieved with a novel transport protein obtainable by modifying the heavy chain of the neurotoxin produced by Clostridium botulinum, wherein
• the protein binds to higher or lower affinity nerve cells than the native neurotoxin
• the protein has increased or decreased neurotoxicity compared to the native neurotoxin; preferably, the neurotoxicity is determined in the hemidiaphragm assay; and or
• the protein has a lower affinity for neutralizing antibodies compared to the native neurotoxin.
• a transport protein which binds to nerve cells with higher or lower affinity than the native neurotoxin produced by C. botulinum.
• a transport protein obtained by modifying the HC of the neurotoxin produced by C. botulinum, wherein the protein having higher or lower affinity than the native neurotoxin binds specifically to nerve cells.
• the transport protein from these cells is taken up by endocytosis.
• a transport protein is also obtained which is obtained by modifying the HC of the neurotoxin formed by C. botulinum, the protein no longer being accessible by exchanging surface-exposed amino acids, in particular at the ganglioside and protein receptor binding pockets of the binding neutralizing antibodies is.
• terms are defined as they are to be understood in the context of the present application.
• “Native neurotoxin is the native neurotoxin of C. botulinum, preferably the botulinum neurotoxin A and / or botulinum neurotoxin B and / or botulinum neurotoxin G from C. botulinum The recombinantly produced botulinum neurotoxin from E.
• the transport protein specifically binds to plasma membrane associated molecules, transmembrane proteins, synaptic vesicle proteins, a synaptotagin family protein or synaptic vesicle glycoproteins 2 (S V2), preferably synaptotagmin I and / or synaptotagmin II and / or SV2A, SV2B or SV2C, more preferably human synaptotagmin I and / or human synaptotagmin II and / or human SV2A, SV2B or SV2C.
• the binding is preferably determined in vitro. Most preferably, the determination is made by using a GST pull-down assay detailed in the Examples.
• the native neurotoxin is the native novotoxin of C. botulinum.
• the native neurotoxin is preferably the botulinum neurotoxin A and / or botulinum neurotoxin B and / or botulinum neurotoxin G from C. botulinum.
• the recombinantly produced botulinum neurotoxin from E. coli which contains, among other things, the amino acid sequence identical to the native botulinum neurotoxin, behaves pharmacologically identically as the native botulinum neurotoxin and is called recombinant botulinum neurotoxin wild type.
• the nerve cells mentioned here are cholinergic motor neurons.
• the neurotoxicity is preferably determined by the hemi-diaphragm assay (HDA) known in the art.
• the neurotoxicity of the muteins can preferably be determined as described by Habermann et al., Naunyn Schmiedeberg's Arch. Pharmacol. 31 1 (1980), 33-40.
• Neurotoxin neutralizing antibodies Botulinum neurotoxin-targeting neutralizing antibodies are known (Göschel H, Wohlfarth K, Frevert J, Dengler R, Bigalke H. Botulinum A toxin therapy: neutralizing and non-neutralizing antibodies-therapeutic consequences, Exp. Neurol 1997 Sep; 147 (1997); l): 96-102.) Neurotoxin neutralizing antibodies have been found to interact particularly with the active sites such as the ganglioside and protein receptor binding pockets within the Hcc domain of the neurotoxin, and in the neurotoxin, the surfaces surrounding the binding pockets are altered by amino acid substitutions Negatively affecting functionality, the neutralizing antibodies lose their binding sites and the mutated neurotoxin is no longer neutralized.
• the amino acid and / or nucleic acid sequence of the heavy chain (HC) of the neurotoxin produced by C. botulinum are generally available from publicly available databases for each of the known serotypes A. to G (see also Table 1).
• Modification here comprises that at least one amino acid is deleted, added, inserted into the amino acid sequence, or at least one amino acid of the native neurotoxin is substituted by another natural or non-naturally occurring amino acid and / or Post-translational modifications include glycosylations, acetylations, acylations, deaminations, phosphorylations, isoprenylations, glycosylphosphatidylinositolations, and other modifications known to the person skilled in the art.
• the HC of the neurotoxin produced by C. botulinum comprises three subdomains, namely the 50 kDa amino terminal translocation domain H N , the subsequent 25 kDa Hc N domain and the carboxyl terminal 25 kDa Hcc domain.
• the H CN and H C c domains are referred to as the Hc fragment.
• the corresponding amino acid sections of the respective subdomains can be seen for the individual serotypes and their variants from Table 1.
• Ganglioside Receptor The HCs of the botulinum neurotoxins have a high affinity for peripheral nerve cells, which are predominantly mediated by the interaction with complex polysial gangliosides - these are glycolipids consisting of more than one sialic acid (Halpern et al. (1995), Curr., Top Microbiol Immunol 195, 221-41, WO 2006/02707) Thus, the LCs bound to them only reach this cell type and become effective only in these cells BoNT / A and B bind only one molecule of ganglioside GTIb.
• the protein receptors are synaptotagmin I and synaptotagmin II.
• the protein receptors are the synaptic vesicles glycoproteins 2 (SV2), preferably SV2A, SV2B and SV2C.
• TMD median transmembrane domain
• the fusion of the synaptic vesicle with the plasma membrane is initiated, whereupon the intraluminal amino terminus of synaptotagmine is extracellularly presented and used as a receptor anchor for BoNT / B and G is available.
• the fourth luminal domain of SV2 isoforms after exocytosis is available extracellularly for interaction with BoNT / A.
• the Hc fragments of BoNT / B and BoNT / G were recombinantly expressed as wild type or with single amino acid substitutions (mutations / substitutions) in the postulated binding pocket in E. coli and isolated.
• the respective GST-synaptotagmin fusion protein with different amounts of the respective Hc fragment of BoNT / B or BoNT / G incubated and carried out a phase separation.
• BoNT / B wild type binds only in the presence of complex gangliosides and synaptotagmin I with transmembrane domain, while synaptotagmin II is bound both with or without transmembrane domain as well as in the presence or absence of complex gangliosides.
• Targeted substitution of amino acids within the protein receptor binding site of BoNT / B significantly increased or decreased the interaction with both synaptotagmin molecules (Figure 1).
• BoNT / G wild type both in the presence and in the absence of complex gangliosides binding to Synaptotagmin I and Synaptotagmin II takes place each with or without transmembrane domain.
• BoNT / B homologous amino acids Within the protein receptor binding site of BoNT / G, the interaction with both synaptotagmin molecules could be significantly increased or attenuated (FIG. 2).
• the potency of the full-length form of BoNT / A, B and G wild types was determined in the HDA by means of a dose-response curve (FIGS. 3 and 6). Subsequently, the potency of the various full-length forms of BoNT / A, B and G single mutants in the HDA was determined (FIG. 6) and related to the potency of the BoNT / B and G wild types by means of an applied power function (FIGS. 4 and 5). , By way of example, the replacement of the amino acids valine 1118 by aspartate or lysine 1192 by glutamate in BoNT / B leads to a drastic reduction of the potency to ⁇ 2%.
• the transport protein has at least 15% higher affinity or at least 15% lower affinity than the native neurotoxin.
• the transport protein has at least 50% higher or lower, more preferably at least 80% higher or lower, and especially at least 90% higher or lower affinity than the native neurotoxin.
• the modification of the HC takes place in the region of the Hc fragment of the given neurotoxin. If the modification comprises a substitution, deletion, insertion or addition, this can be carried out for example by targeted mutagenesis, method These are known to the skilled person.
• the amino acids present in the native neurotoxin are hereby altered either by naturally occurring or non-naturally occurring amino acids. Basically, amino acids are divided into different physicochemical groups.
• the negatively charged amino acids include aspartate and glutamate.
• the positively charged amino acids include histidine, arginine and lysine.
• the polar amino acids include asparagine, glutamine, serine, threonine, cysteine and tyrosine.
• the non-polar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine and tryptophan.
• Aromatic side groups are found in the amino acids histidine, phenylalanine, tyrosine and tryptophan.
• the transport protein is a botulinum neurotoxin serotype A to G.
• amino acid sequences of the native neurotoxins are obtainable from publicly available databases as follows:
• Table 1 Database numbers of the amino acid sequences and subdivision of the seven botulinum neurotoxins.
• the amino acids in the amino acid positions from 867 to 1296 of C. botulinum neurotoxin serotype A, 866 to 1291 of C. botulinum neurotoxin serotype B, 864 to 1291 and 1280, respectively, are preferred for modification C. botulinum neurotoxin serotype Cl, 860 to 1276 or 1285 of C. botulinum neurotoxin serotype D, 843 to 1251 and 1252, respectively, of the C. botulinum and C. butyricum neurotoxin serotype E, respectively 861 to 1274, 862 to 1280 and 1278 and 854 to 1268 of the C. botulinum or
• At least one amino acid in the aforementioned positions be post-translationally modified, and / or added, and / or deleted, and / or inserted, and / or substituted by an amino acid which is either naturally occurring or of non-natural origin.
• the neurotoxin botulinum neurotoxin is serotype A.
• phenylalanine 1255 asparagine 1256, isoleucine 1258 and / or lysine 1260 the botulinum neurotoxin serotype A, protein sequences post-translationally modified, and / or added, and / or deleted, and / or inserted and / or substituted by an amino acid, either naturally occurring or not of natural origin.
• Particularly preferred are the positions asparagine 1196, glutamine 1199, serine 1207, phenylalanine 1255, isoleucine 1258 and / or lysine 1260 of the botulinum neurotoxin serotype A protein sequences.
• the positions serine 1207 substituted by arginine or tyrosine and lysine 1260 substituted for glutamate are preferred.
• the neurotoxin is botulinum neurotoxin serotype B.
• positions valine 1 1 18, tyrosine 1 183, glutamate 1 191, lysine 1 192, glutamate 1245 and tyrosine 1256 of the botulinum neurotoxin serotype B protein sequences are preferred.
• positions tyrosine 1183 and glutamate 1 191, which are replaced by leucine are preferred.
• the neurotoxin is botulinum neurotoxin serotype G.
• positions methionine 1126, leucine 1191, threonine 1199, glutamine 1200, lysine 1250 and tyrosine 1262 of the botulinum neurotoxin serotype G protein sequences are particularly preferred.
• the position tyrosine 1262, which is replaced by phenylalanine, is preferred.
• the transport protein provided in the present invention has an increased or decreased specific affinity of its protein binding domain, in particular to molecules of the synaptotagmine or synaptic vesicle glycoproteins 2 family.
• a further embodiment of the present invention relates to a composition which contains a transport protein according to the invention and at least one intervening molecule (X).
• the intervening molecule may be a small organic molecule, a peptide or a protein; preferably covalently by a peptide bond, ester bond, ether bond, sulfide bond, Disulfide bond or carbon-carbon bond bound to the transport protein.
• the intervening molecule additionally comprises all known therapeutically active substances. Preference is given here cytostatics, antibiotics, antivirals, but also immunoglobulins.
• the protein is a protease which cleaves one or more proteins of the neurotransmitter release mechanism, the protease being selected from the group of neurotoxins selected from the LC of C. botulinum neurotoxins, in particular serotypes A, B, Cl, D, E, F and G or a proteolytically active fragment of the LC of a C. botulinum neurotoxin, in particular a neurotoxin of the serotype A, B, Cl, D, E, F and G, the fragment being at least 0, 01% of the proteolytic activity of the native protease, preferably at least 5%, more preferably at least 50%, especially at least 90%.
• the protease being selected from the group of neurotoxins selected from the LC of C. botulinum neurotoxins, in particular serotypes A, B, Cl, D, E, F and G or a proteolytically active fragment of the LC of a C. botulinum neurotoxin, in particular a neurotoxin of the
• the transport protein and the protease are derived from the same C. botulinum neurotoxin serotype, more preferably the H t M domain of the transport protein and the protease are derived from the C. botulinum neurotoxin serotype A.
• the sequences of the proteases are generally accessible from databases and the database numbers are shown in Table 1. The proteolytic activity of the proteases is determined by means of a substrate cleavage kinetics (see Binz et al. (2002), Biochemistry 41 (6), 1717-23).
• a process for producing the transport protein In a first step, a nucleic acid encoding the transport protein is provided here.
• the coding nucleic acid may be RNA, DNA or mixtures thereof.
• the nucleic acid may also be modified with regard to its nuclease resistance, such as, for example, B. by incorporation of phosphorothioate bonds.
• the nucleic acid can be prepared from an initial nucleic acid be, wherein the starting nucleic acid z. B. is accessible by cloning from genomic or cDNA libraries.
• the nucleic acid can be prepared directly by solid phase synthesis. Suitable methods are known to the person skilled in the art. If it is assumed that an initial nucleic acid, z.
• the nucleic acid is then operably linked to a suitable promoter.
• suitable promoters for expression in known expression systems are known to the person skilled in the art. The choice of the promoter depends on the expression system used for the expression. In general, constitutive promoters are preferred, but inducible promoters are also useful.
• the construct thus produced comprises at least part of a vector, in particular regulatory elements, the vector being selected, for example, from ⁇ derivatives, adenoviruses, baculoviruses, vaccinia viruses, SV40 viruses and retroviruses.
• the vector is preferably capable of expressing the nucleic acid in a given host cell.
• the invention provides host cells that contain the vector and that are suitable for expression of the vector.
• Numerous prokaryotic and eukaryotic expression systems are known in the prior art, the host cells being selected, for example, from prokaryotic cells such as E. coli or B. subtilis, from eukaryotic cells such as S. cerevisiae and P. pastoris.
• prokaryotic cells such as E. coli or B. subtilis
• eukaryotic cells such as S. cerevisiae and P. pastoris.
• higher eukaryotic cells can be used, such as insect cells or mammalian cells, host cells are preferred which, like C. botulinum, have no glycosylation apparatus.
• the nucleic acid encodes the Hc fragment of C. botulinum neurotoxin.
• This nucleic acid contains endonuclease cleavage sites flanking the Hc fragment-encoding nucleic acid, which endonuclease sites are compatible with those of other Hc fragments of C. botulinum neurotoxins to facilitate their easy modular exchange in the permitting the gene encoding the transport protein while retaining the similarity of the amino acid sequence.
• composition according to the invention which contains, in addition to the transport protein, at least one intervening molecule, and this intervening molecule is functionalized with either a carboxy-terminal cysteine or a mercapto group, then it can be carried out in an analogous manner as before described the peptide or the protein are produced recombinantly, for example using binary vectors or by different host cells. If the same host cell is used for the expression of both the transport protein and the peptide or protein, preferably an intermolecular disulfide bond is formed in situ. For more efficient production in the same host cell, the nucleic acid encoding the peptide or protein can also be translated with that of the transport protein in the same reading frame to produce a single-chain polypeptide.
• an intramolecular disulfide bond then preferably forms in situ.
• an amino acid sequence which is either recognized and cleaved specifically by the protease thrombin or a specific endoprotease of the host cell, is inserted at the amino terminus of the transport protein.
• the insert sequence CXXXZKTKSLVPRGSKBXXC (SEQ ID NO: 1), wherein X is any amino acid and Z and B are independently selected from alanine, valine, serine, threonine and glycine, from an endogenous protease of a bacterial host, preferably E. coli is efficiently cleaved in vivo.
• the insertion of the insert sequence between the amino acid sequence of the transport protein and a further peptide or protein therefore has the advantage that subsequent post-processing, such as thrombin, for example, is not required.
• Particularly preferred is the E. co / ⁇ strain E. coli Kl 2.
• the insert sequence is preferably part of a loop containing 18-20, preferably, amino acids.
• a corresponding intermolecular disulfide bond can subsequently be brought about by oxidation processes known to the person skilled in the art.
• the peptide or protein can also be obtained directly by synthesis or fragment condensation. Corresponding methods are known to the person skilled in the art.
• the transport protein and the peptide or protein are subsequently purified.
• the skilled worker known methods are used, such. As chromatography method or electrophoresis.
• Another embodiment of the present invention relates to the pharmaceutical composition containing the transport protein or a composition and optionally a pharmaceutically acceptable carrier, diluent and / or additive.
• the pharmaceutical composition is suitable for oral, intravenous, subcutaneous, intramuscular and topical administration. Intramuscular administration is preferred.
• a dosage unit of the pharmaceutical composition contains about 0.1 pg to 1 mg of transport protein and / or the composition according to the invention.
• the pharmaceutical composition is useful for the treatment of disorders of neurotransmitter release and diseases such as, e.g. As hemifacial spasm, spasmodic torticollis, blepharospasm, spasticity, dystonia, migraine, pain, diseases, the cervical and lumbar spine, strabismus, hyper-salivation, wound healing, snoring and depression suitable.
• Another embodiment of the present invention includes a cosmetic composition containing a transport protein and a cosmetically acceptable carrier, diluent and / or additive.
• the cosmetic composition is suitable for the treatment of hyperhidrosis and facial wrinkles.
• FIG. 1 Investigation of the in vitro binding of the wild-type and mutated BoNT / B Hc fragments on truncated GST-Syt I and GST-Syt II fusion proteins in the presence or absence of complex gangliosides by means of GST pull-down assay.
• FIG. 2 Investigation of the in vitro binding of the wild-type and mutated BoNT / G Hc fragments on truncated GST-Syt I and GST-Syt II fusion proteins in the presence or absence of complex gangliosides by means of GST pull-down assay.
• FIG. 3 Dose-response curve of the BoNT / B and G wild types in the HDA.
• the applied power functions allow a relative comparison of the paralysis times of the single mutants with those of the associated wild types.
• FIG. 4 Decrease and increase of the neurotoxicity of the BoNT / B single mutants in FIG.
• FIG. 5 Decrease and increase of the neurotoxicity of the BoNT / G single mutants in FIG.
• FIG. 6 Dose-response curves of the BoNT / A wild type and the BoNT / A single mutants in the HDA.
• the present invention involves a transport protein (Trapo) which results from modifying the HC of the C. botulinum-produced neurotoxin, preferentially binds specifically to neurons, and is preferentially acquired intracellularly by receptor-mediated endocytosis and from the acidic endosomal compartment into the cytosol of Neurons is translocated.
• This protein is used as a transporter to infect cell-bound proteases and other substances that can not physiologically penetrate the plasma membrane and enter the cytosol of nerve cells.
• the substrates of the proteases are intracellularly localized proteins and peptides involved in transmitter release. After cleavage of the substrates, the specific functions of the neurons are blocked, whereby the cells themselves are not damaged. One of these functions is exocytosis, which effects neurotransmitter release. If the release of transmitters is inhibited, the transmission of signals from cell to cell is blocked. For example, striped muscles are paralyzed when the release of acetylcholine is inhibited at the neuromuscular junction. This effect can be used therapeutically when the transport protein is applied to nerve endings of spastic or dystonic muscles. Other active substances include agents with antiviral activity. Conjugated with the transport protein, they are useful for the treatment of viral infections of the nervous system. The present invention also relates to the use of a transport protein to inhibit the release of neurotransmitters.
• Lower affinity transport proteins bind to, but are not taken up by the nerve cells. These transport proteins are therefore suitable as specific transporters to the nerve cell surface.
• the mode of application and dosage of the neurotoxin according to the invention set forth herein may be selected based on criteria such as the solubility of the selected neurotoxin or the intensity of the pain to be treated.
• the treatment interval for C. botulinum native progenitor toxins A and B averages three to four months. Prolonging this interval would reduce the risk of antibody formation and allow longer duration of treatment with BoNT.
• the increase in LC in the cytosol would extend their degradation time and thus extend the duration of action.
• the transport protein described here has a higher affinity and uptake rate than the native HC.
• Plasmids for the E. coli expression of recombinant Hc fragments of BoNT / B and BoNT / G and the full-length form of BoNT / A, B and G with carboxyl-terminal StrepTag for affinity purification were analyzed by PCR Method with suitable primers, BoNT / A (AAA23262) BoNT / B (AAA23211) and BoNT / G (CAA52275) encoding chromosomal DNA and the expression vector pQe3 (Qiagen AG) generated as the starting vector.
• Truncated variants of rat synaptotagmin I (syt I) (amino acids 1-53, amino acids 1-82) and rat synaptotagmin II (syt II) (amino acids 1-61, amino acids 1-90) were transfected into the GST-encoding vector pGEX- 2T (Amersham Biosciences AB). The nucleic acid sequences of all plasmids were confirmed by DNA sequencing. The recombinant Hc fragments and the VoIl-length form of BoNT were prepared in E. coli strain Ml 5 [pRep4] (Qiagen) during a 10-hour induction at room temperature and on a StrepTactin matrix (IBA GmbH) according to cleaned according to the manufacturer's recommendations.
• the GST fusion proteins obtained from E. coli BL21 were isolated using glutathione immobilized on sepharose beads. Fractions containing the desired proteins were pooled and dialysed against Tris-NaCl-Triton buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, pH 7.2).
• GST fusion proteins (0.12 nmol each) immobilized on 10 ⁇ l GT-Sepharose beads were incubated with Hc fragments (0.1 nmol) in the absence or presence of a bovine brain ganglioside mixture (18% GM1, 55% GDIa, 10% GTIb, 2% other ganglooside, Calbiochem, 20 ⁇ g each) in a total volume of 180 ⁇ l Tris-NaCl-Triton buffer for 2 h at 4 ° C. The beads were collected by centrifugation, the supernatant removed and the separated beads each washed three times with 400 ⁇ l of the same buffer. The washed pellet fractions were boiled in SDS sample buffer and assayed along with the supernatant fractions by SDS-PAGE and Coomassie blue staining.
• BoNT / B wild type only binds in the presence of complex gangliosides and synaptotagmin I with transmembrane domain, whereas synaptotagmin II binds to the transmembrane domain. probably bound with or without transmembrane domain as well as in the presence or absence of complex gangliosides.
• BoNT / G wildtype For the BoNT / G wildtype it has been shown that both in the presence and in the absence of complex gangliosides binding to synaptotagmin I and synaptotagmin II takes place respectively with or without transmembrane domain.
• BoNT / B homologous amino acids within the protein receptor binding site of BoNT / G interaction with both synaptotagmin molecules could be significantly enhanced (YI 262F) and attenuated (Q 1200E), respectively (Figure 2).
• the neurotoxicity of the BoNT / A, B and G muteins was determined as described by Habermann et al., Naunyn Schmiedeberg's Arch. Pharmacol. 311 (1980), 33-40.
• the potency of the full-length form of BoNT / A, B and G wild types was determined in the HDA by means of a dose-response curve (FIGS. 3 and 6).
• the potency of the various full-length forms of BoNT / A, B and G single mutants in the HDA was determined (FIG. 6) and related to the potency of the BoNT / B and G wild types by means of an applied power function (FIGS. 4 and 5).

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