DE19944262A1 - Pharmaceutical composition in the form of a nucleic acid-lipid complex, its production and use in gene therapy - Google Patents

Abstract

The invention relates to a pharmaceutical composition in the form of a nucleic acid lipid complex containing at least one cationic lipid, at least one non-cationic lipid, at least one nucleic acid that codes for a protein used for treating vascular diseases, especially a protein having vasodilatory and/or angiogenic properties, and optionally containing appropriate auxiliary and/or addition agents. According to the invention, the cationic lipid (KL) contains a group derived from cholesterol on which at least one cationic amino group selected from a primary, secondary, tertiary amino group and/or from a quaternary ammonium salt is bound via a connecting group selected from carboxamides and carbamoyls, and via a spacer comprised of a linear or branched alkyl group with 1 to 20 carbon atoms. In addition, the size of the nucleic acid lipid complexes ranges from approximately 300 to 800 nm. The invention also relates to the production of the pharmaceutical composition and to its use in gene therapy.

Description

The present invention relates to a pharmaceutical composition in Form of a nucleic acid-lipid complex containing at least one cationic lipid, at least one non-cationic lipid, at least one Nucleic acid coding for a protein for the treatment of vascular diseases, in particular a protein with vasodilators and / or vasodilators Properties, and optionally suitable auxiliaries and / or additives, wherein the cationic lipid (KL) contains a group derived from cholesterol via a connecting group selected from carboxamides and Carbamoylene, and a spacer consisting of a linear or branched Alkyl group with 1 to 20 carbon atoms at least one cationic Amino group selected from primary, secondary, tertiary amino group and / or a quaternary ammonium salt, and wherein the size of the Nucleic acid-lipid complexes are in a range of approximately 300-800 nm. Furthermore, the present invention relates to the production of pharmaceutical composition and its use in gene therapy.

A number of pharmaceutical compositions are already in the form of a nucleic acid-lipid complex has been developed which is a cationic Lipid and a nucleic acid and optionally other auxiliary and / or Contain additives.

Reszka (WO 96/20208 and DE 196 23 916) developed the cationic Cholesterol derivative 3β- [N- (N, N'-dimethylaminoethane) carbamoyl] cholesterol  (DAC-Chol) and described its use for direct liposomal Gene transfer in vivo. In the preparation of the liposomes described DAC-Chol with dioleylphosphatidylethanolamine (DOPE) as a helper lipid in one molar ratio of 3: 2 used. Epand, R.M. et al. (US 5,283,185) described a method for transferring nucleic acids into cells under Use of cationic lipids with protein kinase C-inhibitory Properties, in particular of the cationic lipid 3β- [N- (N ', N'-dimethyl aminoethane) carbamoyl] cholesterol (DC-Chol), and a co-lipid such as DOPE. An acid addition salt from DC-Chol and its use for transfection of animal cells using a liposomal system has also been described (US 5,753,262). Furthermore, amphipathic vehicles, the polyamine Cholesterol conjugates include for the transfer of nucleic acids into Cells, e.g. B. in gene therapy, proposed (US 5,614,503). A liposomal Composition for the transfer of a DNA molecule, which is a cationic Lipid together with a neutral co-lipid has been described in of which the liposomes have a size of approximately 800 nm (WO 98/17814). Complexes of nucleic acid and cationic liposomes have also been used Achieving organ-specific gene expression in a mammal proposed (US 5,676,954). Szoka, F.C. et al. (US 5,811,406) developed one Transfection method using a lipid-polynucleotide complex, which has been stabilized by the addition of a cryoprotective compound and was then lyophilized. The lyophilized complexes became direct used for transfection without reconstitution. Sorgi, F.L. and Huang, L. (WO 96/27393) described a dry powder formulation which has a Contained lyophilized nucleic acid-liposome complex. The lyophilized Complex can be reconstituted as an aerosol for gene transfer in vitro and used in vivo. In particular, DC-Chol and DOPE were considered Lipids, optionally with a sugar used as a cryoprotective compound. Bischoff, R. (WO 98/08489) described lipid-nucleic acid complexes that a cationic lipid (e.g. DC-Chol), a co-lipid (e.g. DOPE), one stabilizing additive (PEG and derivatives thereof) and a nucleic acid  contained. The resulting particles had a size of 500 nm or fewer. Furthermore, Marshall, J. et al. (WO 98/13026) a Composition containing a cationic amphiphilic molecule Nucleic acid molecule and preferably a co-lipid. Finally Debs, R.J. and Zhu, N. (US 5,827,703) described a liposomal complex for the systemic introduction of genetic material into a mammal, the Complex a cationic lipid associated with cholesterol as a non- cationic lipid.

An example of the preparation of DNA-liposome complexes Phosphatidylcholine, phosphatidylserine and cholesterol and their successful Use in the transfection of vessel walls with the help of Sendai viruses was described in DE 44 11 402. Containing a transfection system an infiltrator catheter, a nucleic acid in the form of a nucleic acid Liposome complex, and possibly suitable auxiliaries and / or additives and its use for the treatment of vascular diseases are from DE 197 29 769 known.

It is easy to see from the aforementioned documents that numerous Efforts have been made to find the most suitable transfection to provide a system for use in gene therapy. This is especially true also for the gene therapy treatment of vascular diseases, such as High blood pressure, arteriosclerosis, stenosis or restenosis.

The task of the work on the present invention was to formulate and a method for the non-viral transfer of nucleic acids into vascular Find cells that efficiently transfer the therapeutic gene into the cells Target cells support local enrichment of a therapeutic protein in the vessel wall with the greatest possible protection of the surrounding tissue accomplished and the commercial production of a lyophilizate in a for enables the appropriate form of distribution.  

It has now surprisingly been found that therapeutic liposomal Formulations with very high efficiency locally in cells of the vessel wall can transfer if you have nucleic acids with suitable lipids and suitable additives and / or auxiliaries to complexes of a defined size formulated and brings these complexes into the target cells of the vessel wall.

An object of the present invention is therefore a pharmaceutical Composition in the form of a nucleic acid-lipid complex containing at least one cationic lipid, at least one non-cationic lipid, one Nucleic acid coding for a protein for the treatment of vascular diseases, in particular a protein with vasodilators and / or vasodilators Properties and possibly other auxiliaries and / or additives, the cationic lipid (KL) a cholesterol-derived, especially lipophilic, Group contains, at which a connection group is selected from Carboxamides and carbamoyls, and a spacer consisting of a linear or branched alkyl group with 1 to 20, preferably 1 to 10, Carbon atoms, at least one cationic amino group selected from primary, secondary, tertiary amino group and / or a quaternary Ammonium salt, and where the size of the nucleic acid lipid Complexes are in a range of approximately 300-800 nm. Particularly good results can be achieved if you have nucleic acid-lipid complexes with a size 350-550 nm for gene transfer, especially in vivo.

The nucleic acid-lipid complexes are usually liposomal complexes. Generally these contain liposomal complexes, especially the nucleic acids, no viral components. With nucleic acid it is usually genomic DNA, cDNA, synthetic DNA, RNA, mRNA, ribozymes, antisense RNA, synthetic peptide nucleic acids and Oligonucleotides, preferably around a cDNA. The nucleic acid can Example can be used in the form of a suitable DNA expression vector (see e.g. DE 44 11 402). For the protein or polypeptide that is used for treatment  of vascular diseases is used, it is preferably one with vasodilator and / or vaso-forming properties. Examples of proteins the isoforms of nitric oxide synthase have vasodilating properties (NOS) and hemoxygenase (HO), while the isoforms of the monocyte Chemoattractant Protein (MCP) have vascular properties. The The family of nitrogen oxide synthases comprises at least three different isoenzymes: The endothelial enzyme (eNOS), the neuronal enzyme (nNOS) and the inducible NOS (iNOS) (see e.g. DE 44 11 402 and DE 197 29 769). The The hemoxygenase family also includes at least three isoenzymes: The Hemoxygenase 1 (HO-1), Hemoxygenase 2 (HO-2), and Hemoxygenase 3 (HO-3) (see e.g. Soares, M.P. et al. (1998) Nature Medicine 4, 1073; Hancock, W. W. et al. (1998) Nature Medicine 4, 1392; Yoshida, T. et al. (1988) Eur. J. Biochem. 171, 457; McCoubrey, W.K. Jr et al. (1992) Arch. Biochem. Biophys. 295, 13; McCoubrey, W.K. Jr et al. (1997) Eur. J. Biochem. 247, 725). To the Isoforms of the monocyte chemoattractant protein include, among others following proteins: MCP-1, MCP-2, MCP-3, MCP-4 and MCP-5 (see e.g. US 5,212,073; US 5,278,287; Ito, W.D. et al. (1997) Circ. Res. 80, 829; Arras, M. (1998) J. Clin. Invest. 101, 40; WO 97/35982 and WO 98/44953). Especially preferred is a nucleic acid for inducible nitric oxide synthase (iNOS), Hemoxygenase 1 (HO-1), the Monocyte Chemoattractant Protein-1 (MCP-1) or a variant thereof, the respective human form is again particularly preferred.

In addition to its vasodilating effect, the iNOS has an antithrombotic effect and antiproliferative effect. Inflammation mediators such as endotoxins lead to the increased expression of this enzyme, which is also called inducible Nitric oxide synthase is referred to (see also DE 44 11 402 and DE 197 29 769). The hemoxygenase isoforms HO-1 and HO-2 are used, among others, in Vascular system expresses and break down heme to biliverdin biliary pigment. This happens with the release of free iron and carbon monoxide (CO). The latter Similar to the nitrogen oxide (NO) generated by the NOS, exercises a vasodilator  and antithrombotic effect. Although the CO formed by the HO-1 Just as NO inhibits smooth muscle cell proliferation, HO-1 induces im In contrast to NOS, no apoptotic cell death. The HO-1 is also in Transplant models as anti-inflammatory and immunoprotective (Hancock et al., supra; and Soares et al., supra). The isoform 1 of the Monocyte Chemoattractant Protein (also Monocyte Chemotactic Protein-1 or JE cytokine), MCP-1, is involved in arteriogenesis, i.e. H. the formation of Collateral vessels from existing arterioles, important. By Infusion of the MCP-1 protein into artificially induced sites of closure Femoral artery in the rabbit animal model (Ito et al., Supra; Arras et al., Supra) achieve the formation of collateral vessels that block the flow of blood into the restore treated limbs.

The present invention also includes variants of the aforementioned proteins. The term “variant” here refers to proteins or polypeptides understood that a sequence homology, in particular a sequence identity of approx. 70%, preferably approx. 80%, in particular approx. 90%, especially approx. 95% of the aforementioned proteins. This also includes Deletions of the protein in the range from about 1-60, preferably from about 1-30, especially from about 1-15, especially from about 1-5 amino acids. Count next to it also fusion proteins that contain the aforementioned proteins. Under Variants are also understood to mean allelic variants that are derived from other cells or Tissues descended. It also means proteins that are produced by come from different individuals. Accordingly, the present includes Invention also nucleic acids for the aforementioned proteins or Encode polypeptides. Examples of such related nucleic acids are Nucleic acids from different human cells or tissues or allelic variants, as well as nucleic acids, from different human Individuals can originate. In a broader sense, one understands one "Variant" of a nucleic acid according to the present invention Nucleic acid that has a homology, in particular a sequence identity of approx.  60%, preferably approx. 75%, in particular approx. 90% and above all approx. 95%. Suitable techniques and methods are available to those skilled in the art Production and mutagenesis of nucleic acids as well as for gene expression and Protein analysis is available (see, e.g., Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Glover, D. M. (1995) DNA cloning: A practical approach, Volume II: Expression systems, IRL Press; Ausubel et al. (1992) Short Protocols in Molecular Biology, John Wiley &Sons; Rees, A.R. et al. (1993) Protein Engineering: A practical approach, IRL Press). The expression "at least one nucleic acid" means according to the present invention that the nucleic acid-lipid complexes too Combinations of more than one nucleic acid can include, the Nucleic acids for both different forms of a therapeutic protein also encode various of the therapeutic proteins described herein can.

In a preferred embodiment, the cationic lipid is around 3β- [N (N'N'-dimethylaminoethane) carbamoyl] cholesterol (see e.g. Epand et al., supra) or 3β- [N- (N, N'-dimethylaminoethane) carbamoyl] cholesterol (DAC- Chol) (see e.g. Reszka, supra).

The at least one non-cationic lipid is usually a lipid selected from at least one phosphatidylcholine, at least a phosphatidylethanolamine and / or cholesterol. Preferably that is Phosphatidylethanolamine is a phosphatidylethanolamine with a chain length of 10-28 carbon atoms, especially dimyristoylphosphatidylethanolamine (DMPE), Dipalmitoylphosphatidylethanolamine (DPPE) and / or Dioleylphosphatidylethanolamine (DOPE), with DOPE being particularly preferred.

A pharmaceutical composition turned out to be particularly advantageous in which the cationic lipid is DAC-Chol and the non-cationic Lipid is DOPE, preferably in a weight ratio of DAC-Chol to  DOPE from approx. 10:90 to approx. 90:10, whereby all weight ratios that between these specified ratios, such as approx. 20:80, 30:70, 40:60, 50: 50, 60: 40, 70: 30 and 80: 20, but especially in between Ratios such as B. 21: 79, 22: 78, 23: 77, 24: 76, 25: 75, 26: 74, 27: 73, 28: 72, 29: 71 and 31: 69, 32: 68, 33: 67, 34: 66, 35: 65, 36: 64, 37: 63, 38: 62 and 39: 61, are included. A weight ratio of DAC chol is particularly preferred to DOPE of approx. 30:70. Alternatively, the ratio of the cationic Lipids to the non-cationic lipid also expressed as a molar ratio become.

The composition according to the invention is made using total lipid cationic lipid and possibly non-cationic lipid to nucleic acid in the Ratio of approx. 1: 1 to approx. 10: 1, in each case based on the weight. The use of a weight ratio of approximately 4: 1 is particularly advantageous up to approx. 5: 1, which has a surprisingly good transfection efficiency in vivo results.

The composition according to the invention can be used as a solution, in particular as a freshly prepared aqueous solution, or as a lyophilisate, which after reconstitution can be used.

The composition according to the invention preferably contains one or several excipients. A means which the Nucleic acid-lipid complexes in both unlyophilized and lyophilized Form stabilized. This can be used as a measure of the stability of the complexes constant size of these complexes can be used. Advantageously if the stabilizing agent is at least one sugar, at least one inorganic salt and / or at least one polyvalent Alcohol. In a broader sense, sugar is also understood here a sugar alcohol such as B. Mannitol. An example of a multivalued Alcohol is polyethylene glycol (PEG). The combination of sucrose as  Sugar and NaCl as the inorganic salt are particularly preferred. The Processes proposed below for producing the process according to the invention For example, compositions provides for this combination of substances by means of an isoosmotic solution in a suitable process step bring in. Those skilled in the art will appreciate the present description further possibilities for the inclusion of the stabilizing agent can be seen his.

The composition according to the invention can also contain at least one Additive included. This is preferably at least one specifically the molecule that recognizes the target cells and / or a gene transfer into the Cell-facilitating molecule. The specific recognition of cells called one also called "targeting". In principle, at least stand for this targeting Two options are available: First, you can use antibodies against Structures on the cell surface such as B. Use receptors that are viral or liposomal vector systems are integrated (Vingerhoeds, H. et al. (1994) Immunmeth 4, 259; Wickham, T.J. et al. (1996) J. Virol. 70, 6831), and other peptides with high binding affinity for receptors on the Use cell surface. Accordingly, the term "a" is used herein Molecule specifically recognizing the target cells "cell-specific or tissue-specific understood binding antibodies or peptides. Such peptides can be in parts a receptor-ligand system can be known or z. B. by means of a Isolate by searching a combinatorial peptide library (Lu, Z. et al. (1995) Biotechnol. 13, 366; U.S. Patent No. 5,635,182; Koivunen, E. et al. (1999) J. Nucl. Med. 40, 883). In addition to the peptides described, there are a number of others Molecules conceivable, also for the specific recognition of a target cell can contribute. In addition to pharmacological agents, this also includes Nucleic acid aptamers that specifically bind structures on the cell surface can (Hicke, B.J. et al. (1996) J. Clin. Invest. 98, 2688). Molecules that Facilitating gene transfer into the cells can work in different ways. On the one hand, they can consist of proteins or peptides attached to a DNA or  a synthetic peptide nucleic acid is present and the transport of the Facilitate nucleic acid in the cell nucleus (Schwartz, B. et al. (1999) Gene Therapy 6, 282; Brandén, L.J. et al. (1999) Nature Biotechnology 17, 784). Furthermore, it can be molecules that release the Improve nucleic acid in the cell cytoplasm (Planck, C. et al. (1994) J. Biol. Chem. 269, 12918; Kichler, A. et al. (1997) Bioconjug. Chem. 8, 213), or to molecules that improve the stability of the nucleic acid in the cell, like that DNA-condensing cationic polymers poly-L-lysine and polyethyleneimine (Lechardeur, D. et al. (1999) Gene Therapy 6, 482).

The present invention further provides a process for the preparation of the pharmaceutical composition according to the invention, which comprises the following steps:

• a) providing a mixture of at least one corresponding cationic lipid (KL) and at least one corresponding non- cationic lipid (NKL), and providing at least one herein defined nucleic acid;
• b) Mixing the mixture of (KL) and (NKL) with the at least one Nucleic acid (N);
• c) if necessary, lyophilization; and
• d) if necessary reconstitute.

By "providing" the starting materials mentioned both previously making a raw material as well as using one already manufactured, possibly commercially available, starting material Roger that. In the latter case, it may be advisable to use the respective Check the concentration information before use and if necessary accordingly adjust.  

In step (ii), the total lipid from (KL) and (NKL) and the Nucleic acid (N) in a ratio of approximately 1: 1 to approximately 10: 1, in each case based on the Weight to be mixed. It can be seen that everything between these Weight ratios of values from the present disclosure are included, e.g. B. approx. 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, especially also all weight ratios, each increasing by 0.1, e.g. B. 4.1: 1, 4.2: 1, etc. Weight ratios of approximately 4: 1 to approximately 5: 1 are particularly preferred. because at these conditions there is a surprisingly high transfection efficiency, especially in vivo.

In a preferred embodiment of the proposed method, in Step (i) the provision of the mixture of (KL) and (NKL) and / or the Provision of the nucleic acid (N) using a stabilizing By means of, in particular at least one sugar, at least one inorganic Salt and / or at least one polyhydric alcohol. As stated above this agent acts in particular adverse effects of a possible Counter lyophilization. The stabilizing agent can be in the form of a Isoosmotic (approx. 300-330 mOsm) solution can be used. A special one a preferred example of a stabilizing agent is a combination of Sucrose as sugar and NaCl as inorganic salt. It is beneficial to do this Use combination in the form of an isoosmotic aqueous solution, for example with a concentration of 100 mM sucrose / 100 mM NaCl or 250 mM sucrose / 25 mM NaCl. Nucleic Acid Lipid Complexes under Use of the latter solutions have been demonstrated particularly good stability and cause efficient transfection in vitro and in vivo.

Another object of the present invention is therefore a pharmaceutical composition according to that proposed herein Procedure can be obtained.  

The present invention further encompasses the use of the pharmaceutical composition according to the invention in gene therapy including combination therapy with pharmacological agents. It may be appropriate to use gene therapy with other therapeutic options To combine approaches such as B. the application of pharmacological Active substances including proteins and / or peptides. For example, a Gene therapy with iNOS can be combined with an application of nitrates, Calcium antagonists and / or beta adrenoreceptor antagonists (with respect to the latter application see Dieterich, H.A. et al. (Ed.) Coronary artery disease, WVG GmbH, Stuttgart, 1993). Treatment of is preferred Vascular diseases, genetic diseases and / or through Gene transfer treatable diseases including their prevention. Their use for the treatment and prevention of is particularly preferred peripheral and / or coronary artery disease. Examples include a. High blood pressure, atherosclerosis, including atherosclerosis of Including grafts as well as the stenosis or restenosis of vessels Vascular grafts, especially coronary heart disease and Myocardial infarction. Treatment / prevention of stenosis of vessels including vascular grafts, restenosis after percutaneous transluminal angioplasty (PTA) of coronary and / or peripheral vessels, deficient blood flow to tissues (this will revascularize the ischemic tissue), coronary artery disease, des Myocardial infarction and / or vascular arteriosclerosis, especially after Transplantation of vessels and / or organs is again special prefers. Another preferred embodiment is the use of the The pharmaceutical composition described herein for the local somatic gene therapy. Thereby, therapeutically effective genes can somatic gene transfer locally in the vessel wall and expressed there be what a compared to the local application of medication Extension of the therapeutic effect enables (see e.g. DE 197 29 769). Local somatic gene therapy using is particularly preferred  a minimally invasive and efficient catheter technology with the targeted individual vessel sections can be transfected in vivo. Is for this purpose an Infiltrator® catheter (see, for example, DE 197 29 769) is particularly preferred.

Usually, the pharmaceutical composition with a Total dose in a range from approx. 0.1 to approx. 20 µg (including all intermediate values), based on the total amount of nucleic acid, administered. In this context it is clear to the person skilled in the art that as "intermediate value" means any value between the specified upper and Lower limits are understood, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, etc .; 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc .; 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, etc.; 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, etc .; 5.0, etc .; 6.0, etc .; 7.0, etc .; 8.0, etc .; 9.0, etc .; 10.0, etc .; 11.0, etc., 12.0, etc .; 13.0, etc .; 14.0, etc .; 15.0, etc .; 16.0, etc., 17.0, etc .; 18.0, etc .; 19.0, etc .; 20.0. It is obvious that the actually used dose, the exact composition, the time and the type the administration as well as the further details of the treatment in view of the present disclosure can be varied. As a suitable animal model can either be the normal domestic pig (Schwartz, R. S. et al. (1990), Circulation 82, 2190; Karas, S.P. et al. (1992) J. Am. Coll. Cardiol. 20, 467) or the so-called mini-pig (Tumbleson, M.E. and Schook, L.B. (1996) Advances in swine in biomedical research, Plenum Press, New York, Vol. 2, 684; see also Unterberg, C. et al. (1995) J. Am. Coll. Cardiol. 26, 1747) can be used. The Results determined in these models can then affect humans be transferred accordingly. Preferably the pharmaceutical Composition with a total dose in a range from approx. 0.5 µg to approx. 10 ug, particularly preferably about 1 ug to about 5 ug, each based on the Total amount of nucleic acid administered.

When using the iNOS as a therapeutically effective gene, especially a vasodilator and antithrombotic effect Achieve antiproliferative effects on the smooth muscle cells of the vascular wall. In  the aforementioned animal model was surprisingly used in vivo in a Total dose of 2 µg DNA found a better transfection efficiency than with a total dose of 10 µg. When using the HO-1 as therapeutic active gene can have a vasodilator and antithrombotic effect achieve. In addition, the HO-1 is used in transplant models described anti-inflammatory and immunoprotective (Hancock et al. (1998), supra; and Soares et al. (1998), supra). Accordingly, the HO-1 can be used as therapeutically active gene in the pharmaceutical described herein Composition also for anti-inflammatory and immune protection of Grafts are used. The introduction of an HO-1 expressing Plasmids, for example in rat aortic grafts, are said to Development of arteriosclerosis in the transplanted vascular sections prevent. In a further step, the expression of the HO-1 can Prevent development of arteriosclerosis in venous grafts which also includes the animal models mouse, rat, pig (see above) and monkey own. The gene transfer can be done here either by means of an Infiltrator® catheter the vascular wall or by perfusion of the vascular grafts. On The central clinical application is thus the prevention of Arteriosclerosis in vascular and organ transplants. There is also an application for Treatment of coronary artery disease and myocardial infarction, as well as of coronary and peripheral occlusive diseases analogous to NOS possible. At Use of MCP-1 as a therapeutically active gene in the herein described pharmaceutical composition can both Arteriogenesis (i.e. the formation of collateral vessels from existing ones Arterioles), as well as angiogenesis (i.e. the formation of new capillaries) be induced. The introduction of an MCP-1 expressing plasmid by means of an Infiltrator® catheter in peripheral or coronary vessels is intended to induce the revascularization of ischemic tissues. This can that of Ito et al. (supra) described rabbit model (especially for peripheral vessels) are used, as well as the animal models described herein of the pig (especially for use in coronary vessels). For  Application is intended for both species of the Infiltrator® catheter. Therapeutic areas of application are therefore especially the coronary Heart disease, peripheral occlusive diseases and myocardial infarction. In a In a particular embodiment, MCP-1 is used together with GM-CSF. "Colony-stimulating factors" (CSFs) are proteins that proliferate and Mediate differentiation of hematopoietic progenitor cells. The Members of this protein family are named after the cell types whose Proliferation or differentiation they stimulate: M-CSF (also called CSF-1, as well as its alternative splice forms such as CSF-4) have a specific effect Macrophages, G-CSF (CSF-3) on granulocytes, while GM-CSF (CSF-2) stimulated both cell types. Another member of this protein family is multi- CSF, known as Interleukin-3. The human GM-CSF cDNA sequence was developed by Wong, G.G. et al. (Science 228, 810, 1985). In the Arteriogenesis causes GM-CSF, possibly with a synergistic effect MCP-1, activation and proliferation of macrophages, causing formation of collateral vessels from arterioles results (WO 99/17798).

Another object of the present invention is therefore a method for therapeutic and / or prophylactic treatment of a subject, the Method of administering an effective amount of the above described pharmaceutical composition comprises.

Finally, another aspect of the invention relates to the use of a isoosmotic composition comprising at least one mono- and / or Disaccharide, and / or at least one polyhydric alcohol and / or at least one inorganic salt for stabilizing nucleic acid lipid Complexes in solution and / or during lyophilization and / or reconstitution. When using the composition for stabilizing nucleic acid Lipid complexes in solution are in particular those in the table below Embodiments 1-5 listed, for stabilization in lyophilization and / or reconstitution particularly preferred embodiments 1-6.  

The use of a combination is particularly advantageous for these purposes setting, the sucrose as disaccharide and sodium chloride as inorganic Contains salt. An example of such an isoosmotic composition (e.g. 300 mOsm) is a combination of sodium chloride in one concentration in a range from approx. 5 mM to approx. 100 mM, in particular 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mM, with an appropriate proportion of sucrose. Of Another is the use of a composition containing mannitol alone or in combination with at least one further mono- and / or disaccharide such as B. sucrose or trehalose preferred for the aforementioned purposes. For example, such an isoosmotic composition (e.g. 300 mOsm) a combination of mannitol in one concentration Range of approx. 10-290 mM, in particular approx. 150-290 mM, and sucrose or Accordingly, trehalose in a concentration in a range of approx. 10- 290 mM, in particular about 10-150 mM, contain.

The following figures and examples are intended to explain the invention in more detail without to limit them to that.  

DESCRIPTION OF THE FIGURES

Fig. 1 shows the expression of iNOS in vitro in smooth muscle cells of the pig: comparison of expression systems DAC 30, DMRIE-C, FuGENE and

Fig. 2 shows the expression of iNOS in vivo in the femoral artery of the pig: comparison of expression systems DAC 30, FuGENE (Fig. 2A), and DMRIE-C (Fig. 2B)

Fig. 3 shows the size of the DAC-30 / DNA complexes as a function of lipid-DNA ratio

Fig. 4 shows the expression of iNOS in the femoral artery of the pig: effect of DNA dose on the expression efficiency in vivo

Fig. 5 shows the expression of the iNOS in vitro in COS-7 cells: expression efficiency in the transfection solutions L11-L15

Fig. 6 shows the expression of iNOS in vivo in the femoral artery of the pig: Comparison of transfection efficiency in solution 11 and 15

Fig. 7 shows the expression of the iNOS in vivo in the femoral artery of the pig: transfection efficiency in solution 15 after lyophilization and reconstitution

FIG. 8 shows the expression of the iNOS in vivo in the A. femoralis of the pig: dependence of the transfection efficiency on the DNA dose in the case of lyophilized / reconstituted complexes ( FIG. 8A: transfection using an Infiltrator® catheter; FIG. 8B: transfection using an infiltrator) ® catheter and implantation of a Wiktor®-i stent)

EXAMPLES I. Material and methods 1. Transfection systems used

• 1. 1.1 Formulations containing DAC-Chol
  The carrier of the liposomal formulation is DAC-30, which is sold by the company GOT (Berlin). DAC-30 ^TM is the trade name for a mixture of DAC-cholesterol and the neutral lipid DOPE in a weight ratio of 30:70 (DAC-Chol: DOPE), which is produced as described in WO 96/20208 and DE 196 23 916. In a corresponding manner, further mixtures of DAC-Chol to DOPE can be produced, such as DAC-40 (40:60) or DAC-50 (50:50). The concentration information was checked before use.
• 2. 1.2 Other transfection systems
  For comparison purposes, DMRIE-C (1,2-dimyristoyl oxypropyl-3-dimethylhydroxyethylammonium bromide) from LifeTechnologies (Rockville, Maryland, USA) and FuGENE ^™ (Boehringer Mannheim) were also used.

2. iNOS expression vectors used

• 1. 2.1 The plasmid pSCMV-iNOS contains the cDNA sequence of the murine iNOS. Its manufacture is described in DE 44 11 402.
• 2. 2.2 The plasmid pcDNA3-HsiNOS contains the cDNA sequence of the human iNOS with a modified 3 'terminus. The human iNOS cDNA was made by circumscribing isolated RNA stimulated human hepatocytes in cDNA. This was in the  Cloning vector pGEM-T (Promega) first inserted the plasmid to construct pGEM-HsiNOS. From this the cDNA with the Restriction endonucleases NotI and ApaI are cut out and inserted into the Expression vector pcDNA3 (Invitrogen) inserted. The so made Plasmid pcDNA3-HsiNOS contains the cDNA for human iNOS, where the 3'-terminal DNA sequence coding for the four C-terminal amino acids MSAL by a DNA sequence coding for an insertion of 20 Amino acids (NPAAMAAGSMRRRALFYSVT) was exchanged.
• 3. 2.3 Plasmid pAH 1 was prepared by cutting out a 0.9 kb Fragments containing the 3 'terminus of the human iNOS cDNA with the Restriction endonuclease SfiI from the plasmid pcDNA3-HsiNOS. This Fragment was replaced by inserting a 0.9 kb PCR fragment, which contained the 3'-terminal, native cDNA sequence of the human iNOS, in the SfiI interface.
• 4. 2.4 Plasmid pAH 9 was cut by cutting a NotI-ApaI fragment that contained the human iNOS cDNA sequence from pAH 1 and insertion of this Fragments obtained in the plasmid pAH 7. The latter was made by Modification of pcDNA3 (Invitrogen) by cutting out a 2 kb comprehensive BbsI-BsmI fragment, filling in the 5 'ends and religation, which introduced the following deletion: parts of the BGH Polyadenylation sequence, the f1 origin of replication, the SV40 Origin of replication, the neomycin resistance gene and parts of the SV40 Polyadenylation sequence. Furthermore, the ampicillin resistance gene containing DNA sequence with the restriction enzyme BspHI cut out and through a PCR fragment with the kanamycin resistance gene from the plasmid pZErO-2 (Invitrogen) replaced.

3. Solutions that can be used to stabilize the lipid-nucleic acid complexes

4. Animal models used

The so-called "Munich mini pig" or "Göttinger mini pig" were used for the studies (see Tumbleson, ME and Schook, LB (1996) supra; Unterberg, C. et al. (1995) supra). The latter experimental animals can be obtained from the University of Göttingen, Institute of Genetics and Animal Breeding and the company Ellegaard Laboratory Pigs (Dalmose, Denmark). By using an injection catheter, transfection to smooth muscle cells in the media cell layer of blood vessels can be localized. The reference value is the DNA dose per injection. If necessary, one can also choose the number of plasmid copies per injection as a reference. For example, in the case of plasmid pAH 9 with a size of 6915 bp, 2 μg correspond to approximately 2.6 × 10 ¹¹ copies. In examples 16 and 17, the Göttingen mini pig was used; in the other experiments the Munich mini pig.

5. Catheter technology used

The Infiltrator® catheter from IVT (San Diego, CA, USA), model DD140015 (see DE 197 29 769) and the Wiktor®-i stent from Medtronic (Minneapolis, MN, USA), Model 6320 used.

6. Preparation and transfection of lipoplexes from DAC-30 and plasmid DNA containing the human iNOS cDNA

• 1. 6.1 Production of the transfection medium
  In the case of solution 15, a solution of 250 mM sucrose and 25 mM NaCl in sterile, pyrogen-free water was prepared. This solution was sterilized in the sterile bench by filtration through a sterile filter with a pore size of 0.2 μm.
• 2. 6.2 Preparation of the DAC-30 solution
  The tubes with DAC-30 were stored unopened at -20 ° C until they were reconstituted. The reconstitution was carried out by adding sterile, pyrogen-free water (final concentration 2 mg / ml). The tubes were closed, shaken at room temperature for 30 min and then mixed for 2 min with a vortex shaker. Before each use, the DAC-30 solution was mixed again with a vortex shaker for 30 seconds.
• 3. 6.3 Preparation of the Lipid-Nucleic Acid Complexes from DAC-30 and DNA
  All necessary work was carried out under sterile conditions. The respective amount of DNA was pipetted into half of the required final volume of transfection solution. Four times the amount of DAC-30 (by weight) was added to the other half of the final volume of transfection solution required. The solutions were homogenized with a vortex shaker. The DAC-30 solution was then slowly added dropwise to the DNA solution. The resulting solution was mixed by pulling it repeatedly into the pipette tip or by rotating the closed reaction vessel.
  A typical example is the following preparation of a DAC-30 / DNA mixture with a lipid-DNA ratio of 4: 1 (w / w) for the administration of a therapeutic dose of 2 µg: 7.5 µl DNA (at a Concentration of 1 µg / µl (corresponding to 7.5 µg) were brought to a final volume of 750 µl by adding transfection solution and homogenized by mixing with the vortex shaker. Four times the amount of DAC-30, ie 30 µg (at a concentration of 2 µg / µl corresponding to 15 µl), was also taken up in 750 µl transfection solution and homogenized by mixing with the vortex shaker. Then 750 µl DAC solution was slowly pipetted dropwise into the 750 µl DNA solution. The resulting solution contained 1.5 ml total volume of lipid-DNA complexes with a DNA concentration of 5 µg / ml. This solution was stable at 4 ° C for at least 48 h and could either be used immediately or lyophilized as described below and reconstituted in 1.5 ml water. 400 µl of this solution could then be injected into the pig's femoral artery as described below, which corresponds to a therapeutic dose of 2 µg DNA.
• 4. 6.4 Scheme and implementation of the lyophilization
  The vessels containing the DAC-30 / DNA lipoplexes were frozen under sterile conditions and freeze-drying was carried out according to the following exemplary scheme. The person skilled in the art can see that freeze drying can also be carried out under different conditions:
  • - 40 hours at -37 ° C shelf temperature and 0.140 mbar pressure.
  • - The temperature of the floor space has risen by 10 ° C / h to 20 ° C.
  • - Post-drying for 2 h at 0.01 mbar.
  • - 6.5 Taking and storing the samples
    The samples were taken under sterile conditions. The lyophilisates were stored at 4 ° C until used.
  • - 6.6 Prepare samples for catheter mediated transfection in vivo
    The lyophilisates (containing a defined amount of DAC-30 / DNA complexes) were reconstituted in 1.5 ml sterile, pyrogen-free water immediately before the operation. The redissolution was checked optically.
  • - 6.7 Transfection of the vessel wall using an infiltrator catheter in vivo (mini pig model)
    A mini pig was intubated, anesthetized, and prepared for surgery, followed by imaging and puncturing of the left carotid artery. Then the guide wire was positioned for a lock, which was followed by the installation of the lock and a dose of 5,000 iU heparin. An overview angiography with a still image was then carried out to show the morphology of the right and left femoral arteries. The prospective transfection sites were determined on the basis of this still image. The guidewire was then positioned in the left femoral artery and the angiography depicted the correct position of the guidewire, and the IVT injection catheter was opened and positioned with subsequent inflation of the balloon of the IVT injection catheter at 3.0 bar. Angiography was then performed to check for complete vascular occlusion. A manual injection of 300 µl or 400 µl of the reconstituted DAC-30 / DNA complexes followed under constant control of the balloon pressure. The catheter was then removed and, optionally, a stent implant at 12 bar for 20 s was positioned exactly at the injection site under angiographic control. When a stent was implanted, 1000-2000 iU heparin was then administered. After a final angiography, all surgical wounds were closed.
  • - 6.8 Evaluation
    The evaluation regarding the transfection efficiency was carried out by immunohistochemical staining of thin sections of the blood vessels, which were taken 3 days after the operation (see DE 197 29 769 with the difference that vessels into which a stent was implanted were embedded in plastic. The layer thickness of the thin sections was 4 µm for plastic preparations and 10 µm for frozen sections). Alternatively, the efficacy was evaluated by ultrasound examination of the test animals over a period of 3-4 weeks and subsequent histochemical processing of thin sections of the removed vessels.

7. Biochemical detection of iNOS

The formation of nitrogen oxide by the iNOS was detected by the detection of nitrite in the cell culture supernatant as follows: 100 .mu.l of the cell culture supernatant to be examined were pipetted into a flat-bottom plate with 96 wells. 50 .mu.l of a 2% sulfanilamide solution in 2.5% H ₃ PO ₄ and 50 .mu.l of a 0.2% naphthylethylenediamine solution in 2.5% H ₃ PO _{4 were} added to the samples. After 5 min incubation at room temperature, the absorption of the samples at 540 nm with a standard series (NaNO ₂ in a concentration of 0 µM, 1 µM, 2.5 µM, 5.0 µM, 10 µM, 20 µM or 40 µM in Cell culture medium) compared.

II. Examples 1. Comparison of liposomal transfection agents for the expression of nitric oxide synthase in vitro: DAC-30, DMRIE-C and FuGENE ^TM

Experiment: Transfection solutions were prepared consisting of cationic liposomes and the plasmid pSCMV-iNOS (murine iNOS-cDNA). The transfection solutions were prepared by mixing a constant dose of 8 μg DNA with the cationic liposomes DAC-30 (1: 5, w / w), DMRIE-C (1: 2, w / v) or FuGENE ^™ (1: 1 , w / v) in BSS buffer (137 mM NaCl, 5.4 mM KCl, 10 mM Tris / Cl pH 7.6). Smooth muscle cells from the pig aorta were cultivated in cell culture dishes with 6 wells each. 500 .mu.l of transfection solution (containing DNA in a concentration of 16 .mu.g / ml) were added to the cells per well, incubated for 1 h at 37.degree. C. and after replacing the solutions with cell culture medium, the cells were further cultivated for 48 h. The amount of nitrite accumulated was then determined from the cell culture supernatant of the transfected cells compared to a control transfection without DNA (see I.7).

Result: The expression of the inducible nitrogen oxide synthase (iNOS) in vitro was dependent on the lipid used. A transfection solution containing DAC-30 / iNOS-DNA resulted in a lower expression of the inducible nitrogen oxide synthase in vitro than transfection solutions containing FuGENE / iNOS-DNA or DMRIE-C / iNOS-DNA (see FIG. 1).

2. Comparison of liposomal transfection agents for the expression of nitric oxide synthase in vivo: DAC-30, DMRIE-C and FuGENE ^TM

Experiment: Transfection solutions were prepared consisting of cationic liposomes and the plasmid pSCMV-iNOS (murine iNOS-cDNA). To prepare the transfection solution, a dose of 10 µg plasmid DNA with DAC-30 in a ratio of 1: 5 (w / w), with DMRIE-C in a ratio of 1: 2 (w / v), or with FuGENE ^™ in a ratio 1: 1 (w / v) mixed in BSS buffer (137 mM NaCl, 5.4 mM KCl, 10 mM Tris / Cl pH 7.6). Two sites of the right and left femoral artery of the pig were transfected in vivo using the Infiltrator® catheter (IVT) with the solutions prepared as described. After a test period of 3 days, the transfected vascular sections were removed. The evaluation of the immunohistochemical staining of thin sections of the removed vessels with a monoclonal antibody against inducible nitrogen oxide synthase was carried out qualitatively in comparison to an untreated control vessel.

Result: Transfection solutions containing DAC-30 / iNOS-DNA showed a significantly better transfection efficiency in vivo than liposome-DNA complexes containing FuGENE ^TM / iNOS-DNA or DMRIE-C / iNOS-DNA. In view of the in vitro data obtained in Example 1, this was a very surprising result (see FIGS . 2.A and 2.B).

3. DAC-30: Expression and toxicity in vitro depending on the lipid DNA ratio

Experiment: Smooth muscle cells from the porcine aorta (see Example 1) were cultivated in cell culture dishes with 6 wells each and transfected with liposome-DNA complexes consisting of DAC-30 and the plasmid pAH 1 (human iNOS cDNA). To prepare the transfection solution (0.9% NaCl, 2 mM CaCl ₂ ), DAC-30 was used at a constant dose of 4 µg DNA (corresponding to a concentration of 8 µg / ml) in a ratio of 2: 1, 4: 1, 5: 1, 6: 1 and 8: 1 mixed. Alternatively, at a constant liposome-DNA ratio of 5: 1, a DNA dose of 2 µg / ml, 4 µg / ml, 8 µg / ml or 16 µg / ml was used to prepare the transfection solution. 500 μl of transfection solution per well were added to the cells, incubated for 1 h at 37 ° C. and, after the solutions had been replaced by cell culture medium, cultured for a further 48 h. After 48 hours, the amount of nitrite accumulated was determined from the cell culture supernatant of the transfected cells compared to a control transfection without DNA in transfection solution (0.9% NaCl, 2 mM CaCl ₂ ) or to untransfected cells (medium) (see I. 7). In parallel, the cytotoxicity of the transfection solution was tested by a vitality test (absorption of neutral red solution).

Result: The expression of the inducible nitric oxide synthase by liposomal Complexes containing DAC-30 / iNOS-DNA depended on the lipid-DNA Ratio in the preparation of the complexes. The expression of the transgene was at a constant dose of 4 µg iNOS plasmid (concentration 8 µg / ml) optimal for a lipid-DNA ratio of 5: 1 liposomal complexes have low cytotoxicity when transfected in vitro. With a suitable liposome-DNA ratio of 5: 1, liposome DNA complexes containing DAC-30 / iNOS-DNA an optimal Expression efficiency at a DNA concentration of 4 µg / ml to 8 µg / ml and low cytotoxicity up to a DNA concentration of 8 µg / ml. (Data Not shown).

4. Complex size depending on the lipid-DNA ratio

Experiment: A transfection solution was prepared consisting of complexes of DAC-30 and the plasmid pAH 9 (human iNOS cDNA) in saline solution (0.9% NaCl / 2 mM CaCl ₂ ). To produce the liposome-DNA complexes, DAC-30 was used in a weight ratio of 2: 1, 3: 1, 4: 1, 5: 1, 6: 1 and 8: 1 to the plasmid DNA at a constant DNA concentration of 6 , 7 µg / ml DNA. The size of the complexes was determined by photon correlation spectroscopy (Zetasizer).

Result: Lipid-DNA mixtures containing DAC-30 / iNOS-DNA formed at a constant DNA concentration of 6.7 µg / ml depending on the lipid-DNA ratio (2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 8: 1) complexes of increasing particle size (see FIG. 3).

5. Complex size depending on the amount of DNA affects the Expression efficiency in vivo

Experiment: Lipid-DNA mixtures were prepared consisting of complexes of DAC-30 and the plasmid pcDNA3-HsiNOS (human iNOS cDNA with modified 3'-terminus) in a saline solution (0.9% NaCl, 2 mM CaCl ₂ ) . To produce the liposome-DNA complexes, DAC-30 was used in a ratio of 5: 1 to the plasmid DNA at a DNA dose of 2 µg (concentration 6.7 µg / ml) or 10 µg DNA (concentration 33.3 µg / ml). Immediately after production, the size of the complexes formed was determined by photon correlation spectroscopy (Zetasizer). With appropriately prepared transfection solutions, two sites of the right and left femoral artery of the pig were transfected in vivo using an Infiltrator ^TM catheter. After a test period of 3 days, the transfected vascular sections were removed. The evaluation of the immunohistochemical staining of thin sections of the removed vessels with a monoclonal antibody against inducible nitrogen oxide synthase was carried out qualitatively in comparison to an untreated control vessel.

Result: The freshly prepared lipid-DNA mixtures containing DAC-30 / iNOS-DNA in a ratio of 5: 1 formed complexes of a smaller particle size at a dose of 2 µg iNOS plasmid (corresponding to a concentration of 6.7 µg / ml) ( approximately 580 nm) than at a dose of 10 µg iNOS plasmid (corresponding to a concentration of 33.3 µg / ml (approximately 1000 nm). When transfected into the vascular wall of the pig's artery in vivo, these mixtures showed better transfection efficiency at a dose of 2 µg iNOS plasmid than at a dose of 10 µg iNOS plasmid The better transfection efficiency of a lower dose in vivo (see FIG. 4) was surprising and correlated with a smaller particle size of the DNA liposome Complexes at this dose.

6. Effectiveness: prevention of neointima formation after transfection of iNOS-DNA / DAC-30

Experiment: Transfection solutions were prepared consisting of DAC-30 and 0.7 µg (corresponding to a DNA concentration of 2.3 µg / ml) of the plasmid pAH 1 (human iNOS cDNA) or the control plasmid pcDNA3 (without cDNA insertion ) in a 5: 1 ratio in a saline solution (0.9% NaCl / 2 mM CaCl ₂ ). Two sites on the right and left femoral artery of the pig were transfected in vivo using the Infiltrator® catheter (IVT) with the solutions prepared as described. A Wiktor®-i stent (Medtronic) was then placed at each transfection site to induce neointima formation. After a test period of 6 weeks, the transfected vessel sections were removed. Thin sections of the removed vessels were evaluated quantitatively by morphometry compared to the control (plasmid pcDNA3) (n = 3).

Result: Containing the expression of the iNOS after transfection of solutions DAC-30 / iNOS DNA in the pig's femoral artery in vivo caused one Reduction of neointima formation (determined as the ratio of neointima to Media) by approx. 20% (data not shown).  

7. Time-dependent stability of lipid-DNA complexes

Experiment: Lipid-DNA mixtures consisting of DAC-30 and the plasmid pAH 9 (human iNOS cDNA) in the ratio 4: 1 and 5: 1 in one increasing DNA concentration of 2.3 µg / ml, 6.7 µg / ml and 13.3 µg / ml in Solution 11 (0.9% NaCl). The size of the resulting liposome DNA Complexes were added immediately after production or after a standing time of 3 hours 4 ° C determined by photon correlation spectroscopy (Zetasizer).

Result: Lipid-DNA mixtures containing DAC-30 / iNOS-DNA formed a lipid-DNA ratio of 4: 1 complexes in a defined Size range from approx. 540 to 700 nm, which also increases with increasing DNA Concentration of 2.3 µg / ml, 6.7 µg / ml and 13.3 µg / ml over a period of Remained constant at 4 ° C for 3 h. This constant particle size is a measure of that Stability of the complexes. In contrast, at a lipid-DNA ratio of 5: 1 the size of the complexes with an increasing amount of DNA of 2.3 µg / ml, 6.7 µg / ml and 13.3 µg / ml over a period of 3 h at room temperature as follows: At 2.3 µg / ml on average by a factor of 2 from approx. 650 nm to approx. 1280 nm, at 6.7 µg / ml by a factor of 3 from approx. 740 nm to approx. 2200 nm and at 13.3 µg / ml by a factor of 2.5 from approx. 940 nm to approx. 2300 nm time-dependent increase in size means that the complexes were unstable. (Data Not shown).

8. Time-dependent stability of lipid-DNA complexes in different Transfection solutions

Experiment: A lipid-DNA mixture consisting of DAC-30 was produced and the plasmid pAH 9 (human iNOS cDNA) in a ratio of 4: 1 in one constant DNA concentration of 5 µg / ml. The production of these liposome DNA complexes took place in different solutions: L 12 (300 mM glucose), L 13 (300 mM sucrose), L 14 (100 mM sucrose, 100 mM NaCl), L 15  (250 mM sucrose, 25 mM NaCl) or L 11 (0.9% NaCl). The size of the Liposome-DNA complexes were either made immediately after or after Storage at 4 ° C for 3 h, 24 h or 48 h by photon correlation Spectroscopy (Zetasizer) determined.

Result: Lipid-DNA mixtures containing DAC-30 / iNOS-DNA formed a lipid-DNA ratio of 4: 1 and a DNA concentration of 5 µg / ml Complexes in a defined size range from approx. 460 to 610 nm. This The complex size remained in the transfection solutions L 13, L 14 and L 15 largely constant over a period of 3 h to 48 h at 4 ° C (approx Factor 1.2 in solution 13, approx. By factor 1.4 in solution 14 or approx. 1.1 in Solution 15), d. H. the complexes formed were stable. DAC-30 / DNA complexes on the other hand, which were produced in L 11, showed over a period of 3 h to 48 h a significant increase in particle size approx. By a factor of 2.5, i.e. H. the complexes formed were unstable. A slight increase in particle size, about a factor of 1.6 was also found in solution 12. (Data not shown)

9. Expression efficiency in vitro in different transfection solutions

Experiment: A transfection solution was prepared consisting of DAC-30 and the plasmid pAH 9 (human iNOS cDNA) in the solutions L 12 (300 mM glucose), L 13 (300 mM sucrose), L 14 (100 mM sucrose, 100 mM NaCl), L 15 (250 mM sucrose, 25 mM NaCl) or L 11 (0.9% NaCl). To prepare the transfection solution, DAC-30 was added in a ratio of 4: 1 to a constant amount of DNA of 2 µg (corresponding to a concentration of 5 µg / ml). The liposome-DNA complexes were transfected into COS-7 cells immediately after production. After 48 hours, the nitrite formed was detected in the cell culture supernatant (see I.7). Average values from 3 independent experiments are shown, the expression level was normalized via the measured values of a neutral red test (see FIG. 5).

Result: The expression of the inducible nitrogen oxide synthase by lipid-DNA mixtures containing DAC-30 / iNOS-DNA was largely independent of the composition of the transfection solution. The solutions L 12, L 13, L 14 and L 15 showed an expression efficiency comparable to L 11 with a constant lipid-DNA ratio of 4: 1 and a dose of 2 µg DNA (see FIG. 5).

10. Transfection efficiency in vivo in solution 15 compared to solution 11

Experiment: Transfection solutions were prepared consisting of DAC-30 and 2 µg of the plasmid pAH 1 (human iNOS cDNA) in a ratio of 5: 1 in solution 15 (250 mM sucrose, 25 mM NaCl) or solution 11 (0.9% NaCl). Two sites on the right and left femoral artery of the pig were transfected in vivo using the Infiltrator ^TM catheter (IVT) with the solutions prepared as described. After a test period of 3 days, the transfected vascular sections were removed. Quantitative evaluation of the immunohistochemical data: plot of the mean values of 5 sections in the center of the transfection site (distance 500 µm each) (n = 2 pigs).

Result: Liposome-DNA complexes containing DAC-30 / iNOS-DNA in L 15 showed a better transfection efficiency in vivo at a dose of 2 µg iNOS plasmid than complexes produced in L 11. The better effectiveness of the transfection solution L 15 in vivo was not to be expected according to the data generated in vitro (see example 10) (see FIG. 6).

11. Short-term stability of DAC-30 / DNA complexes in solution 15

Experiment: A lipid-DNA mixture consisting of DAC-30 was produced and the plasmid pAH 9 (human iNOS cDNA) in a ratio of 4: 1 in solution 15 (250mM sucrose, 25mM NaCl). The DNA concentration was 2.5 µg / ml,  10 µg / ml, 25 µg / ml, or 50 µg / ml. The particle size of the lipid-DNA mixtures was immediately after preparation or reconstitution of the lyophilized Complexes (0 h), and after a standing time of 3 h at 4 ° C Determined photon correlation spectroscopy (Zetasizer).

Result: Lipid-DNA mixtures containing DAC-30 / iNOS-DNA showed a lipid-DNA ratio of 4: 1 and an increasing DNA Concentration of 2.5 µg / ml, 10 µg / ml, 25 µg / ml and 50 µg / ml in solution 15 a constant particle size over a period of 3 h (increase in size less than 15%). This constant particle size over a period of at least 3 h advantageously also remains after lyophilization of the complexes and then reconstituted in sterile water. The Lyophilization therefore has no influence on the short-term stability of the Liposome-DNA complexes. (Data not shown).

12. Long-term stability of DAC-30 / DNA complexes in solution 15

Experiment: A lipid-DNA mixture consisting of DAC-30 was produced and the plasmid pAH 9 (human iNOS cDNA) in a ratio of 4: 1 in solution 15 (250mM sucrose, 25mM NaCl). The DNA concentration was 2.5 µg / ml, 5 µg / ml or 10 µg / ml. By photon correlation spectroscopy (Zetasizer) was the particle size of the complexes produced immediately (0 h), or after one Service life of 3 h, 24 h and 48 h determined. The so produced Transfection solutions contained a total dose of 1 µg, 2 µg or 4 µg Plasmid DNA and were immediately (0 h), or after a standing time of 3 h, 24 h and transfected in COS-7 cells for 48 h. 48 h after transfection accumulated nitrite detected in the cell culture supernatant (see I.7). In parallel the amount of cellular protein was determined (n = 3).

Result: Lipid / DNA mixtures containing DAC-30 / iNOS-DNA showed a constant lipid-DNA ratio of 4: 1 and an increasing DNA  Concentration of 2.5 µg / ml, 5 µg / ml, and 10 µg / ml in solution 15 over a Period of 48 h a constant mean particle size of approx. 480 nm, approx. 500 nm and approx. 510 nm. The transfection of COS-7 cells with these lipid DNA mixtures caused a constant expression of the transgene after a service life of the transfection solution of up to 48 h. Made in solution 15 DAC-30 / DNA complexes showed advantageously over a lifetime of Transfection solution of up to 48 h a constant particle size and Expression efficiency that is maintained even with increasing DNA concentration stayed. (Data not shown).

13. Transfection efficiency in vivo: influence of the DNA dose

Experiment: Transfection solutions were prepared consisting of DAC-30 and 0.5 µg, 1 µg or 2 µg of the plasmid pAH 9 (human iNOS cDNA) or the control plasmid pAH 7 (without cDNA insertion) in a ratio of 4: 1 in solution 15 (250 mM sucrose, 25 mM NaCl). Two sites on the right and left femoral artery of the pig were transfected in vivo using the Infiltrator ^TM catheter (IVT) with the solutions prepared as described. A Wiktor®-i stent (Medtronic) was then placed at each transfection site. After a test period of 3 days, the transfected vascular sections were removed. The immunohistochemical staining of thin sections of the removed vessels with a monoclonal antibody against inducible nitrogen oxide synthase was evaluated quantitatively by morphometry compared to the control (plasmid pAH 7 or untreated vessel).

Result: Liposome-DNA complexes containing DAC-30 / iNOS-DNA in solution 15 showed a better in vivo dose of 2 µg iNOS plasmid Transfection efficiency as 0.5 µg or 1 µg (approx. 43% compared to approx. 31.3% or 13%, based on the share of transfected media in total media). (Data not shown).  

14. Transfection efficiency in vivo in solution 15 after lyophilization and Reconstitution

Experiment: A transfection solution was prepared consisting of DAC-30 and the plasmid pAH 9 (human iNOS cDNA) in a ratio of 4: 1 in solution 15. For transfection of one place on the right or left artery Femoralis of the pig in vivo using an Infiltrator® catheter became fresh prepared (dose 2 µg DNA) or lyophilized and in sterile water reconstituted transfection solution (0.5; 1; and 2 µg DNA) used. A Wiktor®-i stent (Medtronic) was then placed at each transfection site placed. After a test period of 3 days, the transfected vascular sections. Evaluation of immunohistochemical Staining thin sections of the removed vessels with a monoclonal Antibodies against inducible nitric oxide synthase were quantified by Morphometry.

Result: A transfection solution containing DAC-30 / iNOS-DNA in solution 15 (dose of 2 µg DNA) surprisingly showed a 1.5 times better transfection efficiency in vivo than freshly prepared complexes as reconstituted lyophilisate ( Fig. 7; from left to the right: freshly prepared transfection solution (2 µg); control (2 µg); reconstituted transfection solution (0.5; 1; and 2 µg DNA)).

15. Transfection efficiency in vivo with lyophilized / reconstituted com plexing: DNA dose dependence

Experiment: Transfection solutions were prepared consisting of DAC-30 and 0.7 µg, 1 µg, 1.5 µg, 2 µg and 4 µg of the plasmid pAH 9 (human iNOS cDNA) and the control plasmid pAH 7 (without cDNA Insertion) in a ratio of 4: 1. The DNA-liposome complexes were prepared in solution 15, lyophilized and reconstituted in sterile water immediately before the transfection. Two sites on the right and left femoral artery of the pig were transfected in vivo using the Infiltrator ^™ catheter with the solutions prepared as described ( FIG. 8A). The experiment was alternatively carried out with the implantation of a Wiktor®-i stent ( FIG. 8B). The vessels were removed after a test period of 3 days. Quantitative evaluation of the immunohistochemical data of the experiments with and without stent implantation: Percentage of the transfected media from the mean values of 10 µm sections at intervals of 1 mm over the entire transfected vascular section.

Result: Liposome-DNA complexes containing iNOS-DNA / DAC-30 as reconstituted lyophilisate in a dose of 2 µg plasmid DNA showed better transfection efficiency in vivo than in a dose of 0.7 µg, 1 µg, 1.5 µg or 4 µg ( Fig. 8A). The better transfection efficiency at a dose of 2 μg in vivo was not influenced by the implantation of a stent (see FIG. 8B; implantation of a Wiktor®-i stent).

16. Effectiveness in vivo: prevention of neointima formation by transfection of 1 µg iNOS

Experiment: A transfection solution was prepared consisting of DAC-30 and 1 µg of the plasmid pAH 9 (human iNOS cDNA) or the control plasmid pAH 7 (without cDNA insertion) in the ratio 4: 1 in solution 15. The solution was lyophilized and immediately before transfection into one place on the right and the left femoral artery of the pig in vivo using the Infiltrator® catheter in reconstituted sterile water. Subsequently, for induction of the neointima Formation of a Wiktor®-i stent (Medtronic) placed at each transfection site. After a test period of 28 days, the transfected was removed Vessel sections. The evaluation was carried out by intravascular ultrasound Examination of the transfected vascular sections made and evaluated the Reduction of the neointima formed after treatment with the iNOS plasmid in the  Comparison to the control plasmid as the ratio of the plaque area to the total Vascular area (n = 8).

Result: Liposomes / DNA complexes prepared in solution 15 containing one Doses of 1 µg iNOS-DNA / DAC-30 caused after lyophilization and Reconstitution in sterile water reduces the formation of neointima in the Pig restenosis model in vivo by approx. 42%. (Data not shown).

17. Efficacy in vivo: prevention of neointima formation by transfection of 0.25 µg, 0.5 µg or 1 µg iNOS

A transfection solution is prepared consisting of DAC-30 and 0.25 µg, 0.5 µg or 1 µg of the plasmid pAH 9 (human iNOS cDNA) or the Control plasmids pAH 7 (without cDNA insertion) in a ratio of 4: 1 in solution 15. The solution is lyophilized and immediately in one each before transfection The right and left femoral artery of the pig in vivo using Infiltrator® catheter reconstituted in sterile water. Then becomes Induction of neointima formation a Wiktor®-i stent (Medtronic) on each Transfection site placed. After a test period of 28 days, the Removal of the transfected vascular sections. The evaluation is done by intravascular ultrasound examination of the transfected vascular sections carried out and evaluated the reduction of the neointima formed Treatment with iNOS plasmid compared to control plasmid as a ratio the plaque surface to the entire vessel surface.

18. Prevention of graft arteriosclerosis in vivo by using gene therapy HO-1

For the experiment to prevent graft arteriosclerosis, 3- 3.5 kg rabbits used. For surgery, the below sterile conditions are performed, the animals are anesthetized. The  Gene transfer into the donor vessel is accomplished using an Infiltrator® catheter carried out through a lock via the exposed right carotid artery is inserted into the iliac artery using a guide wire. The injection of the HO-1 expression plasmid into the vessel wall of the iliac artery using Infiltrator® catheter takes place at low balloon pressure (approx. 0.6 atm.). there become 100-150 µl transfection solution within approx. 30 seconds, consisting of DAC-30 and an HO-1 expression plasmid in a ratio of 4: 1 in Solution 15 injected into the vessel wall. The seat of the inflated catheter will controlled angiographically. Following the removal of the catheter the transfected vascular segment is removed and placed in the A. iliaca of an allogeneic rabbit. After 2-4 months using histochemistry to investigate whether compared to control treated The emergence of arteriosclerotic changes in the animals reduce implanted vessels (intimal hyperplasia, leukocyte infiltration) leaves.

19. Induction of collateral vessel growth in vivo by gene therapy with MCP-1

For the experiment to induce the growth of collateral vessels Rabbits weighing 3-3.5 kg (New Zealand White rabbits) are used. For the surgical procedure that is performed under sterile conditions anesthetized the animals. The exposed femoral artery is separated by two ligatures tied at a distance of 1.5-2 cm so that the branches of the arteria profunda femoris, the arteria circumflexa femoris lateralis and the arteria circumflexa abdominis remain consistent. An Infiltrator® is used for gene transfer. Catheter is used to pass through the exposed right carotid artery Lock is inserted into the femoral artery using a guide wire. The Injection of the MCP-1 plasmid into the proximal to the ligature Vascular section is carried out using an Infiltrator® catheter at low balloon pressure (approx. 0.6 atm.). 100-150 µl  Transfection solution, consisting of DAC-30 and an MCP-1- Expression plasmid 4: 1 in solution 15 injected into the vessel wall. The seat of the inflated catheter is checked angiographically. in the Following the removal of the catheter, all surgical wounds become locked. After 7 days, angiography is used to examine whether the Compared to control-treated animals, collaterals have increased. With histological methods the proliferation of endothelial cells and smooth muscle cells in the vasculature compared to the treated tissues control-treated animals.

20. Induction of collateral vessel growth in vivo by gene therapy with MCP-1 / GM-CSF

For the experiment to induce the growth of collateral vessels Rabbits weighing 3-3.5 kg (New Zealand White rabbits) are used. For the surgical procedure that is performed under sterile conditions anesthetized the animals. The exposed femoral artery is separated by two ligatures tied at a distance of 1.5-2 cm so that the branches of the arteria profunda femoris, the arteria circumflexa femoris lateralis and the arteria circumflexa abdominis remain consistent. Approx. 7-21 days after vascular occlusion becomes a Gene transfer carried out with plasmid DNA which is used for the expression of MCP-1 or GM-CSF leads in the vessel wall. An Infiltrator® is used for gene transfer. Catheter is used to pass through the exposed right carotid artery Lock is inserted into the femoral artery using a guide wire. The Injection of plasmid DNA in the proximal to the ligature Vascular section using Infiltrator® catheter takes place at low balloon pressure (approx. 0.6 atm.). 100-150 µl Transfection solution consisting of DAC-30 and plasmid DNA in a 4: 1 ratio in solution 15 injected into the vessel wall. The seat of the inflated catheter is checked angiographically. Following the removal of the catheter all surgical wounds are closed. After another 7 days, means  Angiography examines whether compared to control treated animals have increasingly formed collaterals. With histological methods the Proliferation of endothelial cells and smooth muscle cells in the vascular system treated tissues compared to control treated animals.

Claims (27)

1. Pharmaceutical composition in the form of a nucleic acid-lipid complex containing

• a) at least one cationic lipid (KL);
• b) at least one non-cationic lipid (NKL);
• c) at least one nucleic acid (N) coding for a protein for the treatment of vascular diseases, in particular a protein with vasodilating and / or vascular forming properties; and
• d) optionally further auxiliaries and / or additives;

wherein the cationic lipid (KL) contains a group derived from cholesterol, at least one cationic amino group selected from primary via a connecting group selected from carboxamides and carbamoyls and a spacer consisting of a linear or branched alkyl group having 1 to 20 carbon atoms , secondary, tertiary amino group and / or a quaternary ammonium salt,
and wherein the size of the nucleic acid-lipid complexes is in a range of approximately 300-800 nm. 2. The pharmaceutical composition according to claim 1, wherein the size the nucleic acid-lipid complexes in a range of approx. 350-550 nm lies.   3. Pharmaceutical composition according to claim 1 or 2, wherein the Nucleic acid (N) for one of the isoforms of nitric oxide synthase (NOS), the Hemoxygenase (HO), the Monocyte Chemoattractant Protein (MCP), or encodes a variant of one of these proteins. 4. Pharmaceutical composition according to any one of claims 1-3, where at the nucleic acid for the inducible nitrogen oxide synthase (iNOS), the Hemoxygenase-1 (HO-1), the monocyte chemoattractant protein-1 (MCP-1), or a variant thereof, preferably for the respective huma ne form, coded. 5. A pharmaceutical composition according to any one of claims 1-4, where for the cationic lipid (KL) 3β- [N- (N, N'-dimethylaminoethane) - carbamoyl) cholesterol (DAC-Chol) or 3β- [N- (N ', N'-dimethylamino ethane) -carbamoyl] cholesterol (DC-Chol). 6. A pharmaceutical composition according to any one of claims 1-5, where for the non-cationic lipid (NKL) a lipid selected from minde least one phosphatidylcholine, at least one phosphatidylethanol min and / or cholesterol. 7. The pharmaceutical composition according to claim 6, wherein the Phos phatidylethanolamine a diacylphosphatidylethanolamine with a ket is 10-28 carbon atoms, preferably dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE) and / or dioleylphosphatidylethanolamine (DOPE). 8. The pharmaceutical composition according to claim 7, wherein the cationi is lipid (KL) DAC-Chol and the non-cationic lipid (NKL) DOPE is preferably in a weight ratio of DAC-Chol to DOPE from approx. 10:90 to approx. 90:10, particularly preferably from approx. 30:70.   9. A pharmaceutical composition according to any one of claims 1-8, where in the composition using total lipid from (KL) and (NKL) to nucleic acid (N) in a ratio of about 1: 1 to about 10: 1 preferably about 4: 1 or about 5: 1, each based on the weight has been. 10. A pharmaceutical composition according to any one of claims 1-9, where the composition is in the form of a solution or a lyophilisate. 11. Pharmaceutical composition according to one of claims 1-10, the said excipient being a stabilizing agent, in particular at least one sugar, at least one polyhydric alcohol and / or at least one inorganic salt. 12. Pharmaceutical composition according to one of claims 1-11, said additive being at least one specific to the target cells recognizing molecule and / or at least one gene transfer into the Is a cell-facilitating molecule. 13. A method for producing the pharmaceutical composition according to any one of claims 1-12, characterized by the following steps:

• a) providing a mixture of a cationic lipid (KL) according to any one of claims 1-12 and a non-cationic lipid (NKL) according to any one of claims 1-12, and providing a nucleic acid (N) according to any one of claims 1-12 ;
• b) mixing the mixture of (KL) and (NKL) with the nucleic acid (N);
• c) optionally lyophilizing; and
• d) if necessary reconstitute.

14. The method of claim 13, wherein in step (ii) the total lipid (KL) and (NKL) and the nucleic acid (N) in a ratio of approx. 1: 1 to about 10: 1, preferably about 4: 1 or about 5: 1, each based on the Ge important to be mixed. 15. The method according to claim 13 or 14, wherein in step (i) the provision development of the mixture of (KL) and (NKL) and / or the provision of the Nucleic acid (N) using a stabilizing agent, esp special at least one sugar, at least one polyhydric alcohol hauls and / or at least one inorganic salt. 16. The method of claim 15, wherein the stabilizing agent is in the form an isoosmotic aqueous solution is used. 17. Pharmaceutical composition obtainable by the method according to any of claims 13-16. 18. Use of the pharmaceutical composition according to one of the Claims 1-12 or 17 for use in gene therapy Combination therapy with pharmacological agents. 19. Use according to claim 18 for the treatment of vascular diseases, genetic diseases and / or by gene transfer therapier diseases, including their prevention. 20. Use according to claim 18 or 19 for treatment and prevention of peripheral and / or coronary artery disease. 21. Use according to claim 20, wherein the vascular disease is the stenosis of vessels including vascular grafts that cause restenosis after an egg percutaneous transluminal angioplasty (PTA) of coronary arteries  and / or peripheral vessels, a disease due to deficiency blood flow to tissues, coronary artery disease, myocardium farkt, the vascular arteriosclerosis and / or a disease leading to the repulsion leads to vascular and / or organ transplants. 22. Use according to any one of claims 18-21 for the local somatic Gene therapy. 23. Use according to claim 22, wherein the gene therapy using a catheter, in particular an infiltrator catheter. 24. Use according to claim 22 or 23, wherein the pharmaceutical zu composition with a total dose in a range of approximately 0.1-20 µg, preferably about 0.5-10 µg, particularly preferably about 1-5 µg, each based on the total amount of nucleic acid per application is enough. 25. Use of an isoosmotic composition comprising at least least one mono- and / or disaccharide and / or at least one more valuable alcohol and / or at least one inorganic salt for stabilization lization of nucleic acid-lipid complexes in solution. 26. Use of an isoosmotic composition comprising at least least one mono- and / or disaccharide and / or at least one more valuable alcohol and / or at least one inorganic salt for stabilization lization of nucleic acid-lipid complexes during lyophilization and / or reconstitution. 27. Use according to claim 25 or 26, wherein the disaccharide Saccharo se and the inorganic salt is sodium chloride.