WO2001038552A2

WO2001038552A2 - Excipient system that contains coating substances with enzymatically removable side-groups

Info

Publication number: WO2001038552A2
Application number: PCT/EP2000/011055
Authority: WO
Inventors: Hans-Harald Sedlacek; Thomas Kissel
Original assignee: Aventis Pharma Deutschland Gmbh
Priority date: 1999-11-22
Filing date: 2000-11-09
Publication date: 2001-05-31
Also published as: MXPA02004532A; DE19956029A1; JP2003514895A; AU1520201A; EP1235922A2; CA2391922A1; WO2001038552A3

Abstract

The invention relates to an excipient system that can be obtained by (1) optionally mixing a component a2) with a component a1) in a molar ratio [a2):a1)] of 1:1 to 1000:1, preferably between 10:1 and 100:1, (2) mixing the complex resulting from step (1) or the complex a1) alone with a component b1), preferably in a molar ratio b1):a1) + optionally a2) of 1:1 to 1:1000, said mixing ratio being adjusted in such a manner that the net charge of the resulting total complex is preferably neutral or anionic. Component a1) comprises an active substance a1), a binding structure a1) for the binding structures b1) or a2), wherein the active substance a1) may contain the binding structure a1). Component a2) comprises a binding structure a2) for the binding structure a1) and a carrier a2) which may contain the binding structure a2). Component b1) may comprise a binding structure b1) for the binding structure a1), for the active substance a1), for the binding structure a2) or for the carrier a2), a coating substance that may contain the binding structure b1), an enzymatically removable connector, and an anionic side-group. The invention further relates to the production and to the use of the inventive excipient system.

Description

Carrier systems containing coating substances with enzymatically cleavable side groups

Description 5 1. Introduction

Numerous daily materials for active ingredients have so far been developed. These 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.

These 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.

Previous experience with such vectors in gene therapy for a wide variety of 25 diseases, but especially tumor diseases, shows that after administration of a gene therapeutic vector into the circulation of an organism, this vector is eliminated from the circulation in a short time and for binding to the target cells and for Transfection of these target cells is no longer available (Ogris et al. Gene Ther. 6: 595, 1999; Dash et al., Gene Ther. 6: 643, 30 1999; Li et al., Gene Ther. 5: 930, 1998 ; Liu et al. Gene Ther. 4: 517, 1997). This elimination can take place by degradation of the DNA or by rapid deposition of the vectors in the lungs, the liver or the so-called reticuloendothelial system (RES), especially in the spleen and lymph nodes (Zhu et al., Science 261: 209, 1993).

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. In the case of 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.

Experience to date also shows that the coupling or insertion of a target cell-specific ligand into the vector complex does not significantly reduce its rapid elimination after administration into the bloodstream.

Knowing these problems, there is an urgent need for new preparations of vectors which enable vectors to remain in the circuit for as long as possible and not to be prematurely eliminated from the circuit. To reduce the elimination of cationic lipids or cationic polymers in complex with nucleic acid sequences from the bloodstream, polyethylene glycol (Senior et al., Biochim. Biophys. Res. Acta 1062: 77, 1991; Mori et al., FEBS Lett 284: 263 , 1991; Ogris et al., Gene Ther. 6: 595, 1999), vinyl polymers (Torchilin et al., Biochim. Biophys. Res. Acta 1195: 181, 1994) or other amphipatic polymers (Woodle et al., Bioconjugat. Chem. 5: 493, 1994) coupled to the cationic lipids or cationic polymers or with the aid of negatively charged lipids, anionic liposomes into which the

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. 8: 114, 1998; Schoen et al., J. Lip. Res. 8: 485, 1998). Such modifications led, for example, to a stabilization of the vector particle size, inhibited the aggregation of the vectors with themselves or with blood cells, reduced the opsonization of vectors by binding immunoglobulins, complement factors, fibrinogen or fibronectin, protected (adeno) viral vectors from being eliminated by antibodies (Chillon et al., Gene Ther. 5: 995, 1998) and caused an increase in the blood residence time of vectors, a significantly greater accumulation in subcutaneously growing tumors and transduction of the tumor cells (Ogris et al., Gene Ther. 6: 595, 1999).

At the same time, however, a considerable accumulation of the vectors and transduction of the tissue cells in these organs could also be found in the lungs, spleen and liver (Ogris et al., Gene Ther. 6: 595, 1999), so that it can be concluded that for example, the coupling of PEG is an improvement, but is far from being an optimization of the distribution of vectors.

To e.g. To protect against rapid elimination from the body, 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)).

Liposomes that were modified, for example, by glucuronic acid or by dicetyl phosphate (DCP), phosphatidylglycerol or phosphatidylserine, showed a significantly reduced binding to serum proteins and an extended blood retention time as unmodified neutral or cationic liposomes (Oku et al., Biochem. Biophys. Res. Acta 1280, 149 (1996); Purahashi et al., Biol. Pharmac. Bull. 18: 82 (1995); Namba et al., Chem. Pharmac. Bull. 38, 1663 (1990)). Early attempts were made to incorporate glycolipids or glycopeptides into the lipid layer of nanoparticles to produce carrier systems that could be activated by enzymes. A selected enzyme, for example β-glucuronidase, for example released in tumors or introduced into tumors by administration of an anti-tumor antibody-β-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. As a result of this change, 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)).

Although the principle of action could be cleaved by the example of glucuronic acid conjugates by ß-glucuronidase (Sedlacek et al., Contributions to Oncology 43, 132 (1992)), nanoparticles, in the lipid layer of which glucuronic acid conjugates had been inserted, showed considerable structural instability.

The same was observed when incorporating orosomucoid (α1-acidic glycoprotein), an anionic plasma protein containing neuraminic acid, into the lipid layer of liposomes. This incorporation leads to a considerable osmotic sensitivity of the liposomes for water and alkali - as well as alkaline earth ions and to the destruction of these liposomes (Neitchev et al., Mol. Biol. Reports 1 1, 87 (1986); Int. J. Biochem. Cell Biol 29, 689 (1997)).

So far, there are no stable carrier systems containing active substances, the essential components of which are coating substances which carry enzymatically removable anionic side groups, which are crucial for the stability of the carrier system, so that after the removal of these anionic side groups the carrier system disintegrates and the active substances are released. 2. General description of the invention

According to the present invention, 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:

- Containing a component a1)

An active ingredient a1),

• a binding structure a1) for the binding structures b1) or a2), wherein the active ingredient a1) can contain the binding structure a1);

- optionally containing a component a2)

A binding structure a2) for the binding structure a1)

• a carrier a2) which can contain the binding structure a2); and

a component b1) containing • a binding structure b1) for the binding structure a1), for the active ingredient a1), for the binding structure a2) or for the carrier a2),

A coating substance which can contain the binding structure b1),

An enzymatically cleavable connecting part,

• an anionic side group.

Accordingly, component a2) is either contained or not contained in the carrier system.

According to the invention, 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. After the side groups ensuring this anionic to neutral total charge have been split off by enzymes, for example released from tumors or in the context of inflammation, blood coagulation or fibrinolysis or expressed by cells after transfection with genes which code for these enzymes, 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.

The individual components of the carrier system according to the invention and their applications are described in more detail below.

Component a1)

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. 7: 151, 1996; Mercola et al., Cancer Gene Ther. 2: 47, 1995; Frank-Kamenetski, Annu. Rev. Biochem. 64: 65, 1995 and Fraser et al., Exp. Opin. Invest. Drugs 4: 637, 1995. 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. Examples of such 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.

In a particular embodiment of the invention, 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.

To bind component a1) to component a2) or to component b1), component a1) according to the invention has binding groups. These binding groups can be, for example

- anionic charges, for example inserted by nucleic acids, amino acids or by binding of inorganic acids

- Cationic charges, for example inserted by basic amino acids, by amino groups or by binding of inorganic bases

- Lipophilic groups, for example inserted by aromatic amino acids or fatty acids - epitopes for antibodies

- Antigen-binding parts of antibodies

- SH groups

- Fos Leucin Zipper as wild type or mutates as described in German patent application No. 19900743.8 (unpublished) - Jun Leucin Zipper as wild type or mutates as described in German patent application No. 19900743.8. Component a2)

According to this invention, 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).

The complex formation of component a2) with component a1) and / or the binding of component a2) to component b1) can be carried out, for example, by

- cationic charges

- anionic charges

- lipophilic groups

- epitopes - antigen-binding parts of antibodies

- Fos or Jun Leucin Zipper as wild type or mutated as described in German patent application No. 19900743.8.

The choice of component a2) therefore depends on the selection of component a1) and the choice of component b1).

In a particular embodiment of the invention, 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), Thierry 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. 4, 705 (1993), San et al., Human Gene Ther. 4, 781 (1993) or cationic polymers, for example described by Boussif et al., PNAS USA 92, 7292 (1995), Kaneda et al., Science 243, 375 (1989), Keown et al., Methods in Enzymology 185, 527 (1990), Baker et al., Gene Ther. 4, 773 (1997), Fritz et al., Human Gene Ther. 7, 1395 (1996), Wolfert et al., Human Gene Ther. 7, 2123 (1996) and Solodin et al., Biochem. 34, 13537 (1995).

Cationic polymers also include, for example, cationized albumin. The production and use of cationized albumin has been described in patent application EP-A 0 790 312.

In a particular embodiment of this invention, 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.

In a particular embodiment of the invention, 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)

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. Such page groups can be, for example:

- carboxylic acids

- Amino acids

- Sugar, such as glucuronic acid or neuraminic acid bound inorganic acids, such as phosphoric acids or sulfuric acids,

which are eliminated by 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 β-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

- The orosomucoid (α1 -acid glycoprotein), the structure and isolation of which from the blood in detail by Schmidt in: The Plasma Proteins, Ed. Frank Putnam, Academic Press 1975, 183-228.

- Other plasma proteins containing neuraminic acid, as described by Clamp in: The Plasma Proteins, Ed. Frank Putnam, Academic Press 1975, 163-206, in particular those plasma proteins which carry more than 4% of their molecular weight of neuraminic acid, such as, for example, the corticosteroid-binding protein, haptoglobin, hemopexin, α1-antichymotrysin, α-HS-glycoprotein, β2 -Glycoprotein I and ß2-Glycoprσtein II (Clamp 1975) - Glycosphingolipids containing neuraminic acid, such as GM1, GM2, GM3,

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.

In a particular embodiment of this invention, 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.

For the following enzymes, for example, the following enzymatically cleavable connecting parts can be used (Barrett et al., Mammalian Proteases, Academic Press, London (1980), Panchal et al., Nature Biotechnol. 14, 852 (1996); Pigott et al., Ayad et al., The extracellular Matrix, Academic Press (1994); Yoshida et al., Int. J. Cancer 63, 863 (1995), Petersen et al., J. Biol. Chem. 265, 6104 (1990); Cramer et al., J. Urology 156, 526 (1995); Forsgen et al., FEBS Lett. 213, 254 (1987)):

Enzyme substructure C

cleavage

S6 S5 S4 S3 S2 S1 "* S-1 (S-2) SEQ ID NO.

PlasminogenCys Pro Gly Arg Val (Val) 1 activator Gin Gly Arg (lle) 2 Gly Gly Arg

Pro Gly Ly Arg 3 Arg Phe Lys

Prostate - Pro Arg Phe Lys He (lle) 4 specific Tyr (Val) -

Antigen Arg Arg Phe Phe Leu (His) 5 (lle) (Val) Tyr lle Val

Ser Phe Ser He Gin Tyr lle Val 6

Gly Ser Gin Gin Leu Leu lle Val 7

Gly He Ser Ser Gin Tyr lle Val 8

Cathepsine Pro Arg Phe Lys lle (lle) 9 Tyr (Val) Lys Ser Arg Met 10 (lle)

Lys Met Arg Arg 1 1 (lle) lle Arg Arg Arg 12 (lle)

Arg Ala Arg Leu 13 (lle)

Gin Ala Arg Phe 14 (lle)

Lys Leu Arg Leu 15 (lle)

Lys Arg Val (lle) - Lys

Phe Arg -

Stromelysine Gly Gly Gly Ala Gin (Leu) 16

Gin Leu Gly Val Met (Gin) 17

Ala Ala Ala Ser Leu (Lys) 18th

Val Ala Val Ser Ala (Lys) 19

Leu Ala Ala Asn Leu (Arg) 20

Enzyme substructure C

SEQ.ID.NO.

Collagenase Gly Pro Gin Gly lle (Ala) 21

I Gly Pro Gin Gly Leu (Leu) 22

Gly Pro Gin Gly Leu (Ala) 23

Gly lle Ala Gly lle (Thr) 24

II Gly Leu Pro Gly lle (Gly) 25

Gly Phe Pro Gly lle (Gly) 26

III Gly Pro Ala Gly lle (Ser) 27

Gly Pro Ala Gly lle (Ala) 28

VIII

XI Plasminogen Ser Gly Thr Glu (Val) 29 The amino acid positions (S1-S6 and S-1, S-2) were defined according to Schechter and Berger, (Biochem. Biophys. Res. Commun. 27, 157 (1967)).

In a further particular embodiment, 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.

In a particular embodiment, 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)).

For the following intracellular enzymes, for example, the following amino acid sequences can be used as enzymatically cleavable connecting parts:

S1 S-1 SEQ ID NO.

caspases

Caspase 1, 4.5 WEX D 30

Caspase 2,3,7 DEX D 31

Caspase 6,8,9 L / VEX D 32

Caspase 3.9 DEV D 33

LEH D 34

In a further particular embodiment, the intracellular enzyme is a virus-specific protease. For example, retroviruses express an aspartyl protease that is necessary for the production of infectious viruses. Such virus-specific proteases are released by virus-infected cells upon death and decay. For the following 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:

VIRUS PEPTIDE SEQUENCE

S1 S-1 SEQ ID NO. o-MuLV PRSSL Y P ALTP 35

Mo-MuLV TSQA F P LRAG 36

Mo-MuLV MSKL L A TWS 37

Mo-MuLV TQTSL L T LDDQ 38

Mo-MuLV PLQV L T LNIERR 39

Mo-MuLV TSTL L I ENSS 40

Mo-MuLV VQA L V LTQD 41

MPMV PKDI F P VTET 42

BLV PPAI L P IISE 43

EIAV PSEE Y P IMID 44

HTLV-I APQV L P VMHP 45

MMTV LTFT F P WFMRR 46

MMTV SDLV L L SAEARR 47

MMTV DSKA F L ATDW 48

MMTV DELI L P VKRK 49

MMTV VGFA G A MAEAR 50

Analogously, cleavage sequences for proteases expressed by, for example, can be inserted as an enzymatically cleavable connecting part

poliovirus (Hellen et al., J. Virol. 66, 3330 (1992), Mirzayan et al., J. Gen. Virol. 72, 1159 (1991), Pallai et al., J. Biol. Chem. 264, 9738 ( 1989), Kean et al., J. Gen. Virol. 71, 2553 (1990))

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))

- Epstein Barr virus

- Herpes simplex viruses

(Steffy et al., J. Gen. Virol. 76, 1069 (1995), Robertson et al., J. Virol. 70, 4317 (1996), Düanni et al., J. Biol. Chem. 268, 2048 ( 1993), Matusick-Kumar et al., J. Virol. 69, 4347 (1995))

- hepatitis viruses like

* HBV (Hwang et al., Chinese J. Microbiol. Immunol. 24, 71 (1991))

* HAV (Probst et al., J. Virol. 71, 3288 (1997), Martin et al., Virol. 213, 213 (1995), Schultheiss et al., J. Viol. 69, 1727 (1995))

* HCV (Ingallinella et al., Biochem. 37, 8906 (1998), Suzuki et al., J. Gen. Viol. 76, 3021 (1995), Reed et al., J. Virol. 69, 4127 (1995) , Shimizu et al., J. Virol. 70, 127 (1996))

- smallpox virus - cytomegalovirus

(Qiu et al., Nature 383, 275 (1996), La Femina et al., J. Virol. 70, 4819 (1996), Jones et al., J. Virol. 68, 3742 (1994))

- Dengue virus

(Biedrzycka et al., J. Gen. Virol. 68, 1317 (1987))

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. Such 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:

- An anionic charge if component a), a1) or a2) has a cationic charge

- A cationic charge if component a), a1) or a2) has an anionic charge

- A lipophilicity if the component a), a1) or a2) carries lipophilic groups

Epitopes if component a), a1) or a2) contains antigen-binding parts of an antibody

- Antigen-binding parts of an antibody if component a), a1) or a2) bears the corresponding epitopes

- Fos leucine zipper portion of wild type or mutated if component a), a1) or a2) has the corresponding Jun zipper - Jun leucine zipper portion of wild type or mutated if component a) has the corresponding Fos zipper.

In a preferred embodiment of the invention, 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.

Such substances are preferably used which are characterized by good tolerance after administration to humans. coating substances According to the invention are, for example, peptides with 4-20 amino acids, which are polar or terminal

- Contain at least 3 anionic amino acids, such as aspartic acid and / or glutamic acid

- Contain at least 3 cationic amino acids, such as lysine and / or arginine or

- Contain at least 3 lipophilic amino acids, such as tryptophan, tyrosine and / or phenylalanine.

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. For the purposes of the invention, preference is given to ligands which bind to component b1).

Ligands in the sense of the invention can be:

- Multi-functional ligand systems for target cell-specific transfer of nucleotide sequences, described in EP-A 0 846 772

- Single-chain, double-antigen-binding molecules described in EP-A 0 952 218

- specifically cell membrane penetrating molecules described in DE19850987.1, still unpublished or - target cell-specific, multivalent proteins described in DE19910419.0, still unpublished. Reference is expressly made to the patent applications listed here.

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).

For this purpose, 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. However, the ligand can also be conjugated to a lipid as a lipophilic group, for example as described in US Pat. No. 5,662,930. In a particular embodiment of the invention, 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

- In the 1st step, 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

in the second step 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.

As a result, after administration of the carrier system according to the invention in an organism either locally, on the skin, in an organ, a body cavity, in a connective or supporting tissue or in the bloodstream, the residence time is significantly extended, after administration in the bloodstream, for example, up to several hours some days.

Over this long residence time, 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)).

Furthermore and independently of passive targeting, the carrier systems according to the invention which contain a target cell-specific ligand bind to the target cell. In regions in which enzymes have been released (by cell excretion, by zelitod or by expression of a gene introduced into the cell), the anionic side group is split off from the carrier system. As a result, 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.

The preparation and use of the carrier system according to the invention is illustrated in the following examples.

3. Examples to illustrate the idea of the invention

The following examples illustrate how one could practice the present invention.

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

Production of component a1)

The plasmid expression system "pGL3" from Promega which contains the following nucleotide sequences is used as component a1):

- SV40 promoter and enhancer

(Genbank SV40 circular genome; NIDg 965480: nucleotides 5172-294)

- the cDNA for luciferase

- SV40 poly A signal

Using the methods known to the person skilled in the art, the plasmid is introduced into E. coli bacteria, the bacteria are propagated in culture medium and the plasmid is isolated.

Production of components a2)

Low molecular weight polyethyleneimine (PEI-2000) is produced as described in patent application EP-A 0 905 254. For this 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.

Molecular weight determination using light scattering analysis gives 2000 Da. By comparison, the PEI obtained commercially (from Fluka, Neu Ulm) has a molecular weight corresponding to the light scattering analysis of 791 kDa (HMW-PEI).

Production of component b1)

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.

Manufacturing the vector complex

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 μl 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.

Examination of blood retention time in mice

Mice (NMRI) 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 μg of component a1) in the respective complexes per mouse, the suspension medium is physiological saline, the application volume is 250 μl.

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).

For this purpose, 10 μl heparin (1000 lU / ml Novo Nordisk) are added to 100 μl of blood or plasma during the first incubation at 70% in order to quantitatively isolate the plasmid DNA. Samples of the isolated DNA were applied to 0.8% agarose gel and analyzed by Southern blood, as described in detail by Obris et al. 1999 described.

Results While only traces of plasmid DNA can be detected in the blood plasma after administration of the control preparation after 0.5 hours, the animal administered with the complexes according to the invention can be detected in the blood plasma after 0.5 and also after 2 hours, considerable amounts of DNA.

These results show that, as desired, the vector complexes of the invention are characterized by a significant increase in blood residence time.

Figure legend:

1 components a1) and b1) according to the invention

Fig. 2 interaction of components a1) and b1). Fig. 3 components a1), a2) and b1) according to the invention.

Claims

claims

1. Carrier system, available through

(1) optionally mixing a component a2) with a component a1) in the molar ratio [a2): a1)] from 1: 1 to 1000: 1, preferably between 10: 1 and 100: 1 (2) mixture of the from step ( 1) resulting complex or component a1) alone with a component b1), preferably in a molar ratio b1): a1) + optionally a2) of 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,

in which

Component a1)

An active ingredient a1),

• a binding structure a1) for the binding structures b1) or a2), wherein the active ingredient a1) can contain the binding structure a1); contains

Component a2),

A binding structure a2) for the binding structure a1)

• a carrier a2) which can contain the binding structure a2); contains, and

- component b1),

A binding structure b1) for the binding structure a1), for the active ingredient a1), for the binding structure a2) or for the carrier a2),

An envelope substance which can contain the binding structure b1), an enzymatically cleavable connecting part,

• an anionic side group; contains.

2. Carrier system according to claim 1, in which component a2) is mandatory.

3. Carrier system according to one of claims 1 or 2, wherein the bonds between components a1) and a2), a1) and b1) and a2) and b1) by cationic and anionic charges

- by lipophilic groups - by epitope-antibody bonds

- through leucine-zipper interactions and / or

- S-S bridges.

4. Carrier system according to one of claims 1 to 3, in which component a1) is selected from a group comprising a DNA sequence, an RNA sequence, a plasmid, a virus, a peptide, a protein, a glycopeptide, a glycolipid any drug, an enzyme, a radioactive substance, an antibody, a fragment of an antibody containing the antigen-binding site of the antibody, a single-chain, monospecific, bispecific or multi-specific antigen-binding molecule.

5. Carrier system according to one of claims 1 to 4, in which component a2) is selected from a group comprising a cationic lipid, a cationic polymer and a cationic polypeptide and in which component a1) has a negative charge.

6. Carrier system according to one of claims 1 to 4, in which component a2) is a cationic liposome in which component a1) is enclosed.

7. Carrier system according to one of claims 1 to 6, in which the enzymatically cleavable connecting part of component b1) is split off by phospholipases, peptidases, glycosidases, phosphatases, sulfatases or esterases.

8. Carrier system according to one of claims 1 to 7, in which the enzymatically cleavable connecting part of component b1) is split off by β-glucuronidase, neuraminidase, β-galactosidase, plasminogen activator, plasmin, prostate-specific antigen, cathepsin, stromelysin, collagenase or caspases. 10

9. Carrier system according to one of claims 1 to 8, in which the enzymatically cleavable connecting part of component b1) is connected to the covering substance via a spacer which disintegrates after cleaving the connecting part.

15 10. A carrier system according to one of claims 1 to 8 or according to claim 9, wherein the anionic side group of component b1) at least one carboxylic acid, at least one amino acid, preferably aspartic acid and glutamic acid, at least one sugar, preferably glucuronic acid or neuraminic acid and / or at least contains an inorganic acid, preferably as 0 sulfate or phosphate.

11. Carrier system according to one of claims 1 to 10, in which component b1) is a natural polypeptide which carries at least one anionic side group 5 connected via an enzymatically cleavable connecting part.

12. The carrier system according to claim 11, in which the natural polypeptide is selected from a group comprising orosomucoid, corticosteroid-binding protein, haptoglobin, hemopexin, α1-antichymotrypsin, α-HS-0 glycoprotein, β2-glycoprotein I, β2-glycoprotein II.

13. Carrier system according to one of claims 1 to 10, in which component b1) is a glycosphingolipid containing neuraminic acid.

14. The carrier system according to claim 13, in which component b1) is selected from a group comprising GM1, GM2, GM3, GD3, GT3, 0-acetyl-GD3 and O-acetyl-GT3.

15. Use of a carrier system according to one of claims 1 to 14 for the gene therapy treatment of diseases.

17. Use of a carrier system according to claim 15, wherein the disease is selected from the group comprising cancer and inflammation.

18. A method for producing a carrier system according to one of claims 1 to 14, by

(1) optionally mixture of component a2) with component a1) in a molar ratio [a2): a1)] of 1: 1 to 1000: 1, preferably between 10: 1 and 100: 1 (2) mixture of the from step ( 1) resulting complex or the

Component a1) alone with component b1), preferably in a molar ratio b1): a1) + optionally a2) of 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 ,

19. Medicament containing a carrier system according to one of claims 1 to 14 for the treatment of cancer and / or inflammation.