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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2121/00—Preparations for use in therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• Carrier systems containing coating substances with enzymatically cleavable side groups containing coating substances with enzymatically cleavable side groups
• active ingredients include, for example, nucleic acid sequences, peptides, proteins, 10 glycoproteins and glycolipids, as well as pharmaceuticals.
• An essential goal of these carrier materials is to protect the active substance from being broken down in the body and / or from being rapidly excreted.
• Another aim of these carrier materials is to mediate the contact of the active substance with its target cell in which the active substance is supposed to develop its effect.
• carrier materials are particularly important in gene therapy. Viral and non-viral vectors are known. While the carrier materials are the viral envelope proteins for viral vectors, 20 cationic lipids and cationic polymers have been developed as carrier materials for non-viral vectors. With the help of these cationic substances, nucleic acid sequences were complexed and these complexes were administered as such to the organism.
• RES reticuloendothelial system
• the causes of rapid elimination are varied. They can be too large a negative or positive charge, a too large volume or an opsonization of the vector particles by blood proteins.
• viral vectors they can furthermore be the binding of the virus envelope proteins to virus-specific receptors in the organs and / or also antibodies or immune cells, specific for the viruses which bind to the vectors and thereby eliminate them.
• Nucleic acid sequences complexed with cationic lipids or cationic polymers (U.S. Patent No. 4,946,787; U.S. Patent No. 4,245,737; U.S. Patent No. 5,480,463; Heywood and Eanes, Calc. Tissue Int. 40: 149, 1992; Lee and Huang, J Biol. Chem. 271: 8481, 1996; Balicki and Beutler, Blood 88: 3884, 1996; Lucie et al., J. Lip. Res. 8: 57, 1998; Lakkaraju et al., J. Lip. Res. 8: 74, 1998; Turner et al., J. Lip. Res.
• drugs were also packed in liposomes.
• Anionic liposomes were preferably used for this, since they only bind to cells to a small extent and are only eliminated from the body at a slow rate.
• Anionic liposomes are produced in various ways, for example by using substances that incorporate retroviruses into their viral envelope (Schreier et al. 1992, 1993, 1995) or by inserting dicarboxylic acids or by binding sulfuric acid (Ahl et al. 1995; Li and Wu 1999), glucuronic acid or phosphoric acid (Oku et al., Biochem. Biophys. Res. Acta 1280, 149 (1990); Namba et al., Chemical and Pharmaceut. Bullet. 38, 1663 (1990); Hu et al. , Biochem. Biophys. Res. Acta 1299, 252 (1996)).
• DCP dicetyl phosphate
• glycolipids or glycopeptides into the lipid layer of nanoparticles to produce carrier systems that could be activated by enzymes.
• a selected enzyme for example ⁇ -glucuronidase, for example released in tumors or introduced into tumors by administration of an anti-tumor antibody- ⁇ -glucuronidase fusion protein, should split off the sugar (eg glucuronic acid) from the incorporated glycolipids or glycopeptides and thereby increase it of the pH (cationization) and lipophilicity of the nanoparticle.
• the nanoparticle should bind the support to the neighboring cells and fuse or disintegrate with the cell membrane.
• Active substances enclosed in the nanoparticle for example cytostatics, should be released at the site of expression of the activated enzyme (Sedlacek et al., Contributions to Oncology 43, 132 (1992)).
• the carrier system consists of the components shown in FIGS. 1, 2 and 3.
• the invention relates to a carrier system comprising the following components:
• component a2) is either contained or not contained in the carrier system.
• the total charge of the carrier system consisting of components a1), optionally a2) and b1) is neutral or anionic. This total charge ensures that after administration of the carrier system in an organism, preferably in the bloodstream, the residence time of the carrier system in the blood is as long as possible.
• the carrier system becomes cationic and combines with neighboring negatively charged cells and / or disintegrates and thereby releases component a), which, depending on the active ingredient (component a1), either penetrates into the cell and can develop its effect there, or else acts in the blood fluid.
• Component a1) can be an unmodified or modified DNA sequence or an unmodified or modified RNA sequence.
• the nucleotide sequence can perform an anti-DNA (triplex) or anti-RNA (antisense; ribozyme) function or code for an RNA sequence acting in this way or for a protein.
• the nucleotide sequences and their modification can be such that the nucleotide sequence is largely resistant to degradation by DNAsen or RNAsen. Examples of such nucleotide sequences and their modifications are in Breaker, Nature Biotechnol. 15: 427, 1997; Gerwik, Critical Reviews in Oncogenesis 8: 93, 1997; Mukhopadhyay et al., Crit. Rev. Oncogen.
• the DNA sequence can be linear or circular, for example in the form of a plasmid.
• Component a1) can furthermore be a virus, preferably a virus, into which a nucleic acid sequence foreign to the virus has been inserted using the methods known to the person skilled in the art.
• viruses are RTV, AV, AAV, HSV, vaccinia viruses, influenza viruses.
• Such and further examples are from Vile, (Nature Biotechnol. 15: 840, 1997); McKeon et al., (Human Gene Ther. 7: 1615, 1996); Flotte et al., (Gene Ther. 2: 357, 1995); Jolly, (Cancer Gene Ther. 1:51, 1994) and Dubensky et al., (J. Virol. 70: 508, 1996).
• Component a1) can furthermore represent a peptide, a protein, a glycopeptide, a glycolipid, a natural or synthetic medicament or a substance suitable for in vivo or ex vivo detection, such as an enzyme or a radioactive substance.
• component a1) is an antibody, a fragment of an antibody containing the antigen binding site or a single-chain monospecific, bispecific or multispecific antigen-binding molecule, for example prepared as described in European patent application EP-A 0 952 218.
• component a1) to bind component a2) to component b1), component a1) according to the invention has binding groups.
• binding groups can be, for example
• - anionic charges for example inserted by nucleic acids, amino acids or by binding of inorganic acids
• Lipophilic groups for example inserted by aromatic amino acids or fatty acids - epitopes for antibodies
• component a2) consists of a carrier which can form complexes with component a1) and which at the same time has binding groups for component b1).
• component a2) with component a1) and / or the binding of component a2) to component b1) can be carried out, for example, by
• the choice of component a2) therefore depends on the selection of component a1) and the choice of component b1).
• component a2) is at least one cationic lipid and / or at least one cationic polymer, for example
• cationic lipids for example described by Kao et al., Oncology Reports 5: 625 (1998), Liu et al., J. Biol. Chem. 270, 24864 (1995), Feigner, Human Gene Ther. 7, 1791 (1996), Ledley, Human Gene Ther. 6, 1129 (1995), Goyal et al., J. Liposom. Res. 5, 49 (1995), EMS et al., Gene Ther. 4, 226 (1997), Schofield et al., Br. Med. Bull. 51, 56 (1995), Behr, Bioconj. Chem. 5, 382 (1994), Cotten et al., Curr. Opin. Biotechnol.
• Cationic polymers also include, for example, cationized albumin.
• cationized albumin The production and use of cationized albumin has been described in patent application EP-A 0 790 312.
• component a2) is a polyethyleneimine (PEI), in a further particular embodiment of this invention the polyethyleneimine has a molecular weight in a range from 500-20,000 Da and in a further embodiment has an average molecular weight of about 2000 Da and was prepared as described in patent application EP-A 0 905 254.
• PEI polyethyleneimine
• component a2) is liposomes, produced as described, for example, in US Patents 5,252,348, 5,753,258 and 5,766,625. In a further special embodiment of the invention, these liposomes are cationically charged.
• Component b1) represents a coating substance which binds to component a1) or to component a2) via a binding structure and which carries at least one anionic side group connected via an enzymatically cleavable connecting part.
• page groups can be, for example:
• glucuronic acid such as glucuronic acid or neuraminic acid bound inorganic acids, such as phosphoric acids or sulfuric acids,
• enzymes such as phospholipases, peptidases, glycosidases, phosphatases or sulfatases.
• a special subject of the invention are substances which carry glucuronic acids as anionic side groups which can be split off from the residual body of component b1) by ⁇ -glucuronidases.
• Another particular subject of the invention are substances which carry neuraminic acids as anionic side groups which are cleaved from the residual body of component b1) by neuraminidases.
• These substances according to the invention include, for example
• GD3 and GT3 but especially those gangliosides and their derivatives whose neuraminic acids are easily cleaved by neuraminidase, such as GM3, GD3, O-acetyl GD3 and O-acetyl GT3 (Rodriguez et al., J. Lipd Res. 37, 382 (1996 )).
• the enzymatically cleavable connecting part to the anionic side group of component b1) can be any naturally occurring, enzymatic cleavable compound, such as an ester bond, glycosidic bond or peptide bond.
• the enzymatically cleavable connecting part is selected such that it can be cleaved largely specifically by plasminogen activator, plasmin, prostate-specific antigen, cathepsine, stromelysine or collagenase.
• the enzyme which cleaves the enzymatically cleavable connecting part according to the invention is an intracellular enzyme.
• This intracellular enzyme can be a lysosomal enzyme or a cytoplasmic enzyme. Lysosomal enzymes as well as cytoplasmic enzymes are released during inflammation or another disease process in which cells are activated and / or die and decay.
• the cytoplasmic enzyme is a caspase.
• Cleavage sequences for caspases have been described by Cryns and Yuan, (Genes and Developm. 12, 1551 (1998)) and Gross et al., (EMBO J. 17, 3878 (1998)).
• amino acid sequences can be used as enzymatically cleavable connecting parts:
• the intracellular enzyme is a virus-specific protease.
• retroviruses express an aspartyl protease that is necessary for the production of infectious viruses.
• virus-specific proteases are released by virus-infected cells upon death and decay.
• viral enzymes for example, the following amino acid sequences (Menendez et al., Virol. 196, 557 (1993)) can be used as an enzymatically cleavable connecting part:
• cleavage sequences for proteases expressed by, for example, can be inserted as an enzymatically cleavable connecting part
• Influenza viruses Klenk et al., Mol. Recognition in Host Parasite Interactions Vol. 61, Proc. No. 55662 (1991), Hosoga et al., Antiviral Res. 26, A348 (1995))
• Another special subject of the invention are enzymatically cleavable connecting parts which are connected to the coating substance via a spacer.
• the spacer is preferably designed such that it disintegrates after the anionic side group has been split off.
• spacers are described, for example, in the patent application Jacquesy et al., EP-A 0 511 917, to which express reference is made.
• This spacer is, for example, over a by ß-glucuronidase cleavable connecting part with ß-glucuronic acid and connected at its opposite end to a lipophilic substance.
• the binding structure of the coating substance mediates the binding of component b1) to component a1) or a2).
• the binding structure of the coating substance can represent:
• a cationic charge if component a), a1) or a2) has an anionic charge
• the binding structure is identical to the anionic side group which is connected to the coating substance by an enzymatically cleavable connecting part.
• the coating substance can be any peptide, protein, lipid, glycolipid, glycoprotein or any synthetic substance which has the selected property of the binding structure.
• coating substances are, for example, peptides with 4-20 amino acids, which are polar or terminal
• Enveloping substances according to the invention can also be anionic neutral or cationic liposomes prepared as already described for component a2).
• the invention furthermore relates to the addition of the carrier system according to the invention by adding a ligand.
• This ligand according to the invention binds with component a1) or component a2) or component b1) and at the same time has a binding site for the target cell.
• Ligands in the sense of the invention can be:
• the ligand is preferably bound to a lipophilic structure in the carrier system according to the invention.
• This lipophilic structure can be a component of component a1) or a2) or b1).
• the ligand is selected from one of the groups mentioned above to be provided with a lipophilic group.
• These lipophilic groups can be aromatic amino acids which are coupled to the ligand by the method of recombining DNA.
• the ligand can also be conjugated to a lipid as a lipophilic group, for example as described in US Pat. No. 5,662,930.
• the ligand is linked to a fusiogenic peptide and this in turn to a lipophilic group. Examples of fusogenic peptides are described in detail in patent applications EP-A 0 846 772 and DE19850987.1 (not yet published).
• the conjugation of the target cell-specific protein or peptide with a fusiogenic peptide is preferably carried out by expression as a recombinant fusion protein using the methods known to the person skilled in the art.
• the vector according to the invention is produced, for example, from components a1), a2) b1) and the ligand, for example, in such a way that
• component a2) is mixed with component a1) in a molar ratio [a2): a1)] of 1: 1 to 1000: 1, preferably between 10: 1 and 100: 1, below
• component a1) in the complex with component a2) or component a1) alone with component b1) [in the complex with the ligand or alone] is mixed, preferably in a molar ratio b1): a1) + optionally a2) from 1: 1 to 1: 1000, the mixing ratio being adjusted so that the net charge of the resulting overall complex is preferably neutral or anionic.
• Such carrier systems for active substances according to the invention are largely shielded by their component b1), ie their binding and transfection and transduction of cells is largely reduced.
• the residence time is significantly extended, after administration in the bloodstream, for example, up to several hours some days.
• the carrier systems according to the invention accumulate, for example, in the tumor vascular bed due to the so-called "passive targeting" (Unezaki et al., Int. J. Pharmaceutics 114, 11 (1996); Sadzuka et al., Cancer Lett. 127, 99 (1998) and Wunder et al., Int. J. Oncol. 11, 497 (1997)).
• the carrier systems according to the invention which contain a target cell-specific ligand bind to the target cell.
• the anionic side group is split off from the carrier system.
• the carrier system becomes cationic and binds to a cell and / or disintegrates and releases the active ingredient.
• This active ingredient can be, for example, a nucleic acid in complex with a cationic carrier or a cytostatic.
• This nucleic acid sequence can penetrate into the cell with the aid of the cationic carrier and, depending on its composition, develop its action there, ie inhibit, for example, the transcription or translation of a specific gene or a specific RNA, or transduce the cell to code for expression of the RNA or the protein from this nucleic acid sequence.
• the carrier systems according to the invention are therefore preferably suitable for in vivo administration with the aim of diagnosis, prophylaxis or therapy of diseases.
• Example 1 Preparation of a vector complex with a plasmid, a cationic polymer and a coating substance containing side groups which can be split off enzymatically
• plasmid expression system "pGL3" from Promega which contains the following nucleotide sequences is used as component a1):
• the plasmid is introduced into E. coli bacteria, the bacteria are propagated in culture medium and the plasmid is isolated.
• Low molecular weight polyethyleneimine (PEI-2000) is produced as described in patent application EP-A 0 905 254.
• PEI-2000 Low molecular weight polyethyleneimine
• a 10% Ethyleneimine monomer solution in water (5 ml of ethyleneimine monomer + 45 ml of distilled water, dissolution with stirring) with the addition of 1% (0.5 ml) of concentrated hydrochloric acid (37 ° C.) as catalyst for 4 days at 50 ° C., evaporated and dried under vacuum at room temperature.
• the molecular weight determination is carried out by means of laser scattered light measurement (Wyatt Dwan DSP light scattering photometer) at 633 nm after direct injection into a K5 measuring cell.
• the molar masses are determined on the basis of the calibration constants determined in toluene and the known sample weight.
• orosomucoid As an enveloping substance, orosomucoid is used, purified from human blood serum, as described by Schmidt in: The Plasma Proteins, ed. Frank Putnam, Academic Press 1975, 183-228.
• Plasmids (component a1) are suspended in a concentration of 10 9 plasmids / ml of physiological saline. Component a1) is then added in portions (component a2) PEI-2000 (1 mg / ml physical saline with 1N HCl to pH 7.4) until the complexes formed (component a1 + a2) are completely cationized (pH between 7.5 and 12, preferably 10).
• the cationization is determined in the agarose shift assay, in which 50 ⁇ l aliquots are applied to an approximately 0.5 cm thick gel of 1% (w / v) agarose and in Tris-EDTA buffer pH 7.5 to 80 mV Is developed for 2 hours. The location of the DNA was visualized at 254 nm after reaction with ethidium bromide.
• the cationic complexes from components a1) + a2) are then suspended in an excess of component b1) and incubated at 4 ° C. for at least 48 hours. In this excess, complexes form from the components a1) + a2) + b1), which have a neutral to slightly anionic charge. This charge is checked in the agarose shift assay and should be in the range between pH 4 and 7.
• the vector complex according to the invention [component a1) + a2) + b1)] can then be used.
• the vector complex component a) + b1) is used as a control.
• mice are injected with the complexes according to the invention [component a1) + a2) + b1)] or, as a control, complexes with components a1) + a2) into the tail vein.
• the dose is 50 ⁇ g of component a1) in the respective complexes per mouse, the suspension medium is physiological saline, the application volume is 250 ⁇ l.
• the animals are anesthetized and bled 30 minutes and 2 hours after the injection.
• the collected blood is mixed with sodium citrate (final concentration 25 mM) and the blood plasma is separated from the blood cells by centrifugation (10 min, 1000 g).
• the DNA is isolated from the whole blood or from the blood plasma using the QIAamp Tissue Kit (Qiagen, Hilden).
• heparin 1000 lU / ml Novo Nordisk
• 10 ⁇ l heparin 1000 lU / ml Novo Nordisk
• Fig. 2 interaction of components a1) and b1).
• Fig. 3 components a1), a2) and b1) according to the invention.

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• 1999
  • 1999-11-22 DE DE19956029A patent/DE19956029A1/de not_active Withdrawn
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