EP1216027A2 - Pharmaceutical composition in the form of a nucleic acid lipid complex, the production thereof and its 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

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

The present 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 coding for a protein for the treatment of vascular diseases, in particular a protein with vasodilating and / or vascular forming properties , and optionally suitable auxiliaries and / or additives, the cationic lipid (KL) containing a group derived from cholesterol, via which a connecting group selected from carboxamides and carbamoyls, 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 is in a range of about 300-800 nm. The present invention further relates to the production of the pharmaceutical composition and its use in gene therapy.

A number of pharmaceutical compositions in the form of a nucleic acid-lipid complex have been developed which contain a cationic lipid and a nucleic acid and, if appropriate, further auxiliaries and / or additives.

Reszka (WO 96/20208 and DE 196 23 916) developed the cationic cholesterol derivative 3j8- [N- (N, N'-dimethylaminoethane) carbamoyl] cholesterol (DAC-Chol) and described its use for direct liposomal gene transfer in vivo. DAC-Chol with dioleylphosphatidylethanolamine (DOPE) was used as helper lipid in a molar ratio of 3: 2 in the preparation of the liposomes described. Epand, RM et al. (US 5,283,185) described a method for transferring nucleic acids into cells using cationic lipids with protein kinase C-inhibitory properties, in particular the cationic lipid 3j8- [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), and a co-lipid such as DOPE. An acid addition salt of DC-Chol and its use for the transfection of animal cells by means of a liposomal system has also been described (US Pat. No. 5,753,262). Furthermore, amphipathic vehicles comprising polyamine-cholesterol conjugates have been proposed for the transfer of nucleic acids into cells, for example in gene therapy (US Pat. No. 5,614,503). A liposomal composition for the transfer of a DNA molecule, which comprises a cationic lipid together with a neutral co-lipid, has been described, in which the liposomes have a size of approximately 800 nm (WO 98/17814). Complexes of nucleic acid and cationic liposomes have also been proposed to achieve organ-specific gene expression in a mammal (US Pat. No. 5,676,954). Szoka, FC et al. (US 5,811,406) developed a transfection method using a lipid-polynucleotide complex, which was stabilized by adding a cryoprotective compound and then lyophilized. The lyophilized complexes were used directly for transfection without reconstitution. Sorgi, FL and Huang, L. (WO 96/27393) described a dry powder formulation which contained a lyophilized nucleic acid-liposome complex. After reconstitution, the lyophilized complex can be used as an aerosol for gene transfer in vitro and in vivo. In particular, DC-Chol and DOPE were used as lipids, optionally with a sugar as a cryoprotective compound. Bischoff, R. (WO 98/08489) described lipid-nucleic acid complexes comprising a cationic lipid (eg DC-Chol), a co-lipid (eg DOPE), a stabilizing additive (PEG and derivatives thereof) and a nucleic acid contained. The resulting particles were 500 nm or less in size. Furthermore, Marshall, J. et al. (WO 98/13026) propose a composition comprising a cationic amphiphilic molecule, a nucleic acid molecule and preferably a co-lipid. Finally, Debs, RJ and Zhu, N. (US 5,827,703) described a liposomal complex for the systemic introduction of genetic material into a mammal, the complex comprising a cationic lipid in combination with cholesterol as the non-cationic lipid.

An example of the production of DNA-liposome complexes from phosphatidylcholine, phosphatidylserine and cholesterol and their successful use in the transfection of vessel walls with the help of Sendai viruses has been described in DE 44 11 402. A transfection system containing an infiltrator catheter, a nucleic acid in the form of a nucleic acid-liposome complex, and, if appropriate, suitable auxiliaries and / or additives and their use for the treatment of vascular diseases are known from DE 197 29 769.

It is readily apparent from the aforementioned documents that numerous efforts have been made to provide the most suitable transfection system for use in gene therapy. This applies in particular to 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 find a formulation and a method for the non-viral transfer of nucleic acids into vascular cells, which supports an efficient transfer of the therapeutic gene into the target cells and a local accumulation of a therapeutic protein in the vessel wall the greatest possible protection of the surrounding tissue and enables the commercial production of a lyophilisate in a form suitable for distribution. It has now surprisingly been found that therapeutic liposomal formulations can be transferred locally into cells of the vessel wall with very high efficiency if nucleic acids with suitable lipids and suitable additives and / or auxiliaries are formulated into complexes of a defined size and these complexes into the target cells of the Introduces the vessel wall.

The present invention therefore relates to a pharmaceutical composition in the form of a nucleic acid-lipid complex, comprising at least one cationic lipid, at least one non-cationic lipid, a nucleic acid coding for a protein for the treatment of vascular diseases, in particular a protein with vasodilator and / or vessel-forming properties and, if appropriate, further auxiliaries and / or additives, the cationic lipid (KL) containing a group derived from cholesterol, in particular lipophilic, at which a connecting group selected from carboxamides and carbamoyls and a spacer consisting of a linear or branched alkyl group having 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 wherein the size of the nucleic acid-lipid complexes in a range of c a. 300-800 nm. Particularly good results can be achieved if nucleic acid-lipid complexes with a size of approximately 350-550 nm are used for gene transfer, in particular in vivo.

The nucleic acid-lipid complexes are usually liposomal complexes. In general, these liposomal complexes, especially the nucleic acids, do not contain any viral components. The nucleic acid is usually genomic DNA, cDNA, synthetic DNA, RNA, mRNA, ribozymes, antisense RNA, synthetic peptide nucleic acids and oligonucleotides, preferably a cDNA. The nucleic acid can be used, for example, in the form of a suitable DNA expression vector (see, for example, DE 44 1 1 402). For the protein or polypeptide that is used for treatment is used by vascular diseases, it is preferably one with vasodilator and or vascular properties. Examples of proteins with vasodilating properties are the isoforms of nitrogen oxide synthase (NOS) and hemoxygenase (HO), while the isoforms of monocyte chemoattractant protein (MCP) have vascular properties. 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, for example, DE 44 11 402 and DE 197 29 769). The family of hemoxygenases also includes at least three isoenzymes: hemoxygenase 1 (HO-1), hemoxygenase 2 (HO-2), and hemoxygenase 3 (HO-3) (see, for example, Soares, MP et al. (1998) Nature Medicine 4, 1073; Hancock, WW et al. (1998) Nature Medicine 4, 1392; Yoshida, T. et al. (1988) Eur. J. Biochem. 171, 457; McCoubrey, WK Jr et al. (1992) Arch. Biochem. Biophys. 295, 13; McCoubrey, WK Jr et al. (1997) Eur. J. Biochem. 247, 725). The isoforms of the monocyte chemoattractant protein include the following proteins: MCP-1, MCP-2, MCP-3, MCP-4 and MCP-5 (see, for example, US 5,212,073; US 5,278,287; Ito, WD et al. (1997 ) Circ. Res. 80, 829; Arras, M. (1998) J. Clin. Invest. 101, 40; WO 97/35982 and WO 98/44953). A nucleic acid which codes for the inducible nitrogen oxide synthase (iNOS), the hemoxygenase 1 (HO-1), the monocyte chemoattractant protein-1 (MCP-1) or a variant thereof is particularly preferred, the particular human form in turn being particularly preferred ,

In addition to its vasodilating effect, the iNOS has an antithrombotic and antiproliferative effect. Inflammation mediators such as endotoxins lead to the increased expression of this enzyme, which is also referred to as inducible nitrogen oxide synthase (see also DE 44 11 402 and DE 197 29 769). The hemoxygenase isoforms HO-1 and HO-2 are expressed, among other things, in the vascular system and break down heme to biliverdin, a bile pigment. This happens with the release of free iron and carbon monoxide (CO). The latter, like the nitrogen oxide (NO) generated by NOS, exerts a vasodilator and antithrombotic effect. Although the CO formed by the HO-1, like NO, inhibits the proliferation of smooth muscle cells, in contrast to the NOS, HO-1 does not induce apoptotic cell death. HO-1 has also been described 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 called Monocyte Chemotactic Protein-1 or JE-Cytokin), MCP-1, is important in arteriogenesis, ie the formation of collateral vessels from existing arterioles. By infusing the MCP-1 protein into artificially induced occlusions of the femoral artery in the rabbit animal model (Ito et al., Supra; Arras et al., Supra), the formation of collateral vessels can be achieved which restore the blood flow to the treated extremities.

The present invention also includes variants of the aforementioned proteins. The term “variant” is understood here to mean proteins or polypeptides which have a sequence homology, in particular a sequence identity of approximately 70%, preferably approximately 80%, in particular approximately 90%, especially approximately 95% of the aforementioned Own proteins. This also includes deletions of the protein in the range from about 1-60, preferably from about 1-30, in particular from about 1-15, especially from about 1-5 amino acids. In addition, this also includes fusion proteins that contain the aforementioned proteins. Variants are also understood to mean allelic variants that derive from other cells or tissues. It also means proteins that come from different individuals. Accordingly, the present invention also encompasses nucleic acids which code for the aforementioned proteins or polypeptides. Examples of such related nucleic acids are nucleic acids from different human cells or tissues or allelic variants, as well as nucleic acids that can come from different human individuals. In a broader sense, a “variant” of a nucleic acid according to the present invention means a nucleic acid which has a homology, in particular a sequence identity of approx. 60%, preferably about 75%, in particular about 90% and above all about 95%. Suitable techniques and processes for the production and mutagenesis of nucleic acids as well as for gene expression and protein analysis are available to the person skilled in the art (see, for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Glover, DM ( 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, AR et al. (1993) Protein Engineering: A practical approach, IRL Press). According to the present invention, the expression “at least one nucleic acid” means that the nucleic acid-lipid complexes can also comprise combinations of more than one nucleic acid, the nucleic acids both for different forms of a therapeutic protein and for various of the therapeutic proteins described herein Can encode proteins.

In a preferred embodiment, the cationic lipid is 3 / - [N (N ¹ N'-dimethylaminoethane) carbamoyl] cholesterol (see for example Epand et al., Supra) or 3 3- [N- (N, N'-dimethylaminoethane) carbamoyl] cholesterol (DAC-Chol) (see, for example, Reszka, supra).

The at least one non-cationic lipid is usually a lipid selected from at least one phosphatidylcholine, at least one phosphatidylethanolamine and / or cholesterol. The phosphatidylethanolamine is preferably a phosphatidylethanolamine with a chain length of 10-28 carbon atoms, in particular dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE) and / or

Dioleylphosphatidylethanolamine (DOPE), with DOPE being particularly preferred.

A pharmaceutical composition in which the cationic lipid is DAC-Chol and the non-cationic was found to be particularly advantageous

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 lie between these specified ratios, such as approx. 20:80, 30:70, 40:60, 50:50, 60:40, 70 : 30 and 80:20, but in particular also intermediate conditions, such as 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. A weight ratio of DAC-Chol to DOPE of approximately 30:70 is particularly preferred. Alternatively, the ratio of the cationic lipid to the non-cationic lipid can also be expressed as a molar ratio.

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

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

The composition according to the invention preferably contains one or more auxiliaries. Particularly preferred is an agent that stabilizes the nucleic acid-lipid complexes in both unlyophilized and lyophilized form. The constant size of these complexes can be used as a measure of the stability of the complexes. The stabilizing agent is advantageously at least one sugar, at least one inorganic salt and / or at least one polyhydric alcohol. In a broader sense, a sugar is also understood to mean a sugar alcohol such as mannitol. An example of a polyhydric alcohol is polyethylene glycol (PEG). The combination of sucrose as Sugar and NaCl as the inorganic salt are particularly preferred. The method proposed below for producing the compositions according to the invention, for example, provides for this combination of substances to be introduced by means of an isoosmotic solution in a suitable method step. In view of the present description, the person skilled in the art will see further possibilities for taking up the stabilizing agent.

The composition according to the invention can further contain at least one additive. This is preferably at least one molecule which specifically recognizes the target cells and / or a molecule which facilitates gene transfer into the cells. The specific recognition of cells is also known as "targeting". There are at least two options available for this targeting: Firstly, antibodies against structures on the cell surface, such as receptors, can be used, which are integrated into viral or liposomal vector systems (Vingerhoeds, H. et al. (1994) Immunmeth 4, 259; Wickham, TJ et al. (1996) J. Virol. 70, 6831), and secondly, peptides with high binding affinity for receptors on the cell surface can be used. Accordingly, the term “a molecule which specifically recognizes the target cells” is understood to mean cell- or tissue-specific binding antibodies or peptides. Such peptides can be known as parts of a receptor-ligand system or can be isolated, for example, by searching a combinatorial peptide bank (Lu, Z. et al. (1995) Biotechnol. 13, 366; US Patent No. 5,635,182; Koivunen, E . et al. (1999) J. Nucl. Med. 40, 883). In addition to the peptides described, a number of other molecules are conceivable, which can also contribute to the specific recognition of a target cell. In addition to pharmacological agents, this also includes nucleic acid aptamers that can specifically bind structures on the cell surface (Hicke, BJ et al. (1996) J. Clin. Invest. 98, 2688). Molecules that facilitate gene transfer into 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 facilitate the transport of the nucleic acid into the cell nucleus (Schwartz, B. et al. (1999) Gene Therapy 6, 282; Branden, LJ et al. (1999) Nature Biotechnology 17, 784) , Furthermore, they can be molecules which improve the release of the nucleic acid into the cytoplasm of the cell (Planck, C. et al. (1994) J. Biol. Chem. 269, 12918; Kichler, A. et al. (1997) Bioconjug. Chem. 8, 213), or molecules that improve the stability of the nucleic acid in the cell, such as the 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:

(i) 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 nucleic acid defined herein; (ii) mixing the mixture of (KL) and (NKL) with the at least one nucleic acid (N);

(iii) optionally lyophilizing; and (iv) reconstitute if necessary.

The “provision” of the starting materials mentioned here means both the preceding production of a starting material and the use of an already produced, possibly commercially available, starting material. In the latter case, it may be advisable to check the respective concentration information before use and adjust it if necessary. In step (ii), the total lipid from (KL) and (NKL) and the nucleic acid (N) are usually mixed in a ratio of about 1: 1 to about 10: 1, in each case based on the weight. It can be seen that all of the weight ratios between these values are included in the present disclosure, for example approximately 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9 : 1, in particular also all weight ratios, each increasing by a value of 0.1, for example 4.1: 1, 4.2: 1, etc. Weight ratios of approximately 4: 1 to approximately 5: 1 are particularly preferred, since a surprisingly high transfection efficiency, in particular in vivo, can be determined at these conditions.

In a preferred embodiment of the proposed method, the mixture of (KL) and (NKL) and / or the provision of the nucleic acid (N) is provided in step (i) using a stabilizing agent, in particular at least one sugar, at least one inorganic salt and / or at least one polyhydric alcohol. As stated above, this agent counteracts, in particular, adverse effects of a possible lyophilization. The stabilizing agent can be used in the form of an isoosmotic (approx. 300-330 mOsm) solution. A particularly preferred example of a stabilizing agent is a combination of sucrose as sugar and NaCl as an inorganic salt. It is advantageous to use this 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 which have been produced using the latter solutions have a particularly good stability and bring about efficient transfection in vitro and in vivo.

The present invention therefore furthermore relates to a pharmaceutical composition which can be obtained by the process proposed here. The present invention furthermore relates to the use of the pharmaceutical composition according to the invention in gene therapy, including a combination therapy with pharmacological active ingredients. It can be useful to combine gene therapy with other therapeutic approaches, such as the application of pharmacological agents including proteins and / or peptides. For example, gene therapy with iNOS can be combined with an application of nitrates, calcium antagonists and / or ß-adrenoreceptor antagonists (for the latter application see Dieterich, HA et al. (Ed.) Coronary Heart Disease, WVG GmbH, Stuttgart, 1993). The treatment of vascular diseases, genetically caused diseases and / or diseases which can be treated by gene transfer, including their prevention, is preferred. Their use for the treatment and prevention of peripheral and / or coronary vascular diseases is particularly preferred. Examples include high blood pressure, arteriosclerosis, including arteriosclerosis of transplants, and the stenosis or restenosis of vessels, including vascular grafts, in particular also of 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, insufficient blood flow to tissues (the aim is to revascularize the ischemic tissue), coronary artery disease, myocardial infarction and / or vascular arteriosclerosis, especially after transplantation of vessels and / or organs, is again particularly preferred. Another preferred embodiment is the use of the pharmaceutical composition described herein for local somatic gene therapy. Thereby, therapeutically active genes can be transferred locally into the vessel wall via a somatic gene transfer and expressed there, which enables an extension of the therapeutic effect compared to the local application of medication (see, for example, DE 197 29 769). Local somatic gene therapy using is particularly preferred a minimally invasive and efficient catheter technology with which individual vessel sections can be transfected in vivo. For this purpose a Inf_ltrator ^® catheter (see eg DE 197 29 769) is particularly preferred.

The pharmaceutical composition is usually administered with a total dose in a range from about 0.1 to about 20 μg (including all values in between), based on the total amount of nucleic acid. In this connection it is clear to the person skilled in the art that the “intermediate value” is understood to mean any value between the specified upper and lower limits, 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 actual dose used, the exact composition, the time and the mode of administration as well as the further details of the treatment can be varied in view of the present disclosure. As a suitable animal model, either the normal domestic pig (Schwartz, RS et al. (1990), Circulation 82, 2190; Karas, SP et al. (1992) J. Am. Coll. Cardiol. 20, 467) or the so-called. Minischwein (Tumbleson, ME and Schook, LB (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 be transferred to humans accordingly. The pharmaceutical composition is preferably administered with a total dose in a range from approximately 0.5 μg to approximately 10 μg, particularly preferably approximately 1 μg to approximately 5 μg, in each case based on the total amount of nucleic acid.

When using the iNOS as a therapeutically active gene, in addition to a vasodilator and antithrombotic effect, an antiproliferative effect on the smooth muscle cells of the vascular wall can be achieved. In In the animal model mentioned above, a better transfection efficiency was surprisingly found in vivo with a total dose of 2 μg DNA than with a total dose of 10 μg. When the HO-1 is used as a therapeutically active gene, a vasodilator and antithrombotic effect can be achieved. In addition, HO-1 is described in transplant models as anti-inflammatory and immunoprotective (Hancock et al. (1998), supra; and Soares et al. (1998), supra). Accordingly, the HO-1 as a therapeutically active gene in the pharmaceutical composition described herein can also be used for the inhibition of inflammation and immune protection of transplants. The introduction of a HO-1-expressing plasmid, for example in rat aortic grafts, is intended to prevent the development of arteriosclerosis in the transplanted vascular sections. In a further step, the expression of HO-1 can prevent the development of arteriosclerosis in venous grafts, for which the mouse, rat, pig (see above) and monkey animal models are also suitable. The gene transfer can either take place in the vascular wall using an Infiltrator ^® catheter or by perfusion of the vascular grafts. A central clinical area of application is the prevention of arteriosclerosis in vascular and organ transplants. It can also be used to treat coronary heart disease and myocardial infarction, as well as coronary and peripheral occlusive diseases, analogous to NOS. When MCP-1 is used as a therapeutically active gene in the pharmaceutical composition described herein, both arteriogenesis (ie the formation of collateral vessels from existing arterioles) and angiogenesis (ie the formation of new capillaries) can be induced. The introduction of an MCP-1-expressing plasmid by means of an Infiltrator ^® catheter into peripheral or coronary vessels is intended to induce the revascularization of ischemic tissues. For this, the Ito et al. (supra) described rabbit model (especially for peripheral vessels) are used, as well as the animal models of the pig described here (especially for use in coronary vessels). to Application is intended for both species of the infiltrator catheter. Therapeutic areas of application include coronary heart disease, peripheral occlusive diseases and myocardial infarction. In a particular embodiment, MCP-1 is used together with GM-CSF. "Colony-stimulating factors" (CSFs) are proteins that mediate the proliferation and 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) has a specific effect on macrophages, G-CSF (CSF-3) on granulocytes, while GM-CSF (CSF-2) stimulates both cell types. Another member of this family of proteins is multi-CSF, known as interleukin-3. The cDNA sequence of the human GM-CSF was by Wong, GG et al. (Science 228, 810, 1985). In arteriogenesis, GM-CSF, possibly in a synergistic effect with MCP-1, causes the activation and proliferation of macrophages, which results in the formation of collateral vessels from arterioles (WO 99/17798).

The present invention therefore further provides a method for the therapeutic and / or prophylactic treatment of a subject, the method comprising administering an effective amount of the pharmaceutical composition described above.

Finally, a further aspect of the invention relates to the use of an 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 Lyophilization and / or reconstitution. When the composition is used to stabilize nucleic acid-lipid complexes in solution, the embodiments 1-5 listed in the table below are particularly preferred, and the embodiments 1-6 are particularly preferred for stabilization in the case of lyophilization and / or reconstitution. The use of a composition which contains sucrose as the disaccharide and sodium chloride as the inorganic salt is particularly advantageous for these purposes. An example of such an isoosmotic composition (for example 300 mOsm) is a combination of sodium chloride in a concentration in a range from approximately 5 mM to approximately 100 mM, in particular 5, 10, 15, 20, 25, 30, 35, 40 , 45 or 50 mM, with an appropriate proportion of sucrose. Furthermore, the use of a composition containing mannitol alone or in combination with at least one further mono- and / or disaccharide such as sucrose or trehalose is preferred for the aforementioned purposes. For example, such an isoosmotic composition (eg 300 mOsm) can be a combination of mannitol in a concentration in a range from about 10-290 mM, in particular about 150-290 mM, and sucrose or trehalose in a concentration in a range from about 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 restricting it to them. DESCRIPTION OF THE FIGURES

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

FIG. 2 shows the expression of the iNOS in vivo in the femoral artery of the pig: comparison of the expression systems DAC-30, FuGENE (FIG. 2A) and DMRIE-C (FIG. 2B). FIG. 3 shows the size of the DAC-30 / DNA complexes depending on the lipid-DNA ratio

4 shows the expression of the iNOS in the femoral artery of the pig:

Influence of the 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 L1 1-L15. FIG. 6 shows the expression of the iNOS in vivo in the femoral artery of the pig: comparison of the 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 femoral artery of the pig: dependence of the transfection efficiency on the DNA dose in lyophilized / reconstituted complexes (FIG. 8A: transfection using an Infiltrator ^® catheter; FIG. 8B: transfection using an Infϊltrator ^® catheter and implantation of a Wiktor ^® -i- stents)

EXAMPLES

I. Material and methods

1. Transfection systems used

1.1 Formulations containing DAC-Chol

The carrier of the liposomal formulation is DAC-30, which is available from G.O.T. (Berlin) is distributed. DAC-30 ™ is the trade name for a mixture of DAC cholesterol and the neutral lipid DOPE im

Weight ratio 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.

1.2 Other transfection systems

DMRIE-C (1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium bromide) from LifeTechnologies was also used for comparison purposes

(Rockville, Maryland, USA) and FuGENE ™ (Boehringer Mannheim).

2. iNOS expression vectors used

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 The plasmid pcDNA3-HsiNOS contains the cDNA sequence of the human iNOS with a modified 3 'terminus. The human iNOS cDNA was prepared by transcribing isolated RNA from stimulated human hepatocytes into cDNA. This was in the Cloning vector pGEM-T (Promega) inserted to first construct the plasmid pGEM-HsiNOS. The cDNA with the restriction endonucleases Notl and Apal was cut out of this and inserted into the expression vector pcDNA3 (Invitrogen). The plasmid pcDNA3-HsiNOS thus produced contains the cDNA for the human iNOS, the 3 '-terminal DNA sequence coding for the four C-terminal amino acids MSAL being replaced by a DNA sequence coding for an insertion of 20 amino acids (NPAAMAAGSMRRRALFYSVT) , 2.3 Plasmid pAH 1 was prepared by cutting out a 0.9 kb fragment containing the 3 'terminus of the human iNOS cDNA with the

Restriction endonuclease Sfil 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, into the Sfil site. 2.4 Plasmid pAH 9 was obtained by cutting out a NotI-Apal fragment, which contained the human iNOS cDNA sequence, from pAH 1 and inserting this fragment into the plasmid pAH 7. The latter was prepared by modifying pcDNA3 (Invitrogen) by cutting out a 2 kb BbsI-BsmI fragment, filling in the 5 'ends and religation, which introduced the following deletion: parts of the BGH-

Polyadenylation sequence, the fl replication origin, the SV40 origin of replication, the neomycin resistance gene and parts of the SV40 polyadenylation sequence. Furthermore, the DNA sequence containing the AmpiciUinresistance gene was cut out with the restriction enzyme BspHI and replaced by a PCR fragment with the kanamycin resistance gene from the plasmid pZErO-2 (Invitrogen). 20 -

3. Solutions which 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, M.E. and Schook, L.B. (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 record the number of plasmid copies per injection as

Select reference size. For example, in the case of plasmid pAH 9 with a size of 6915 bp, 2 μg correspond to approximately 2.6 × 10 ⁿ copies. In examples 16 and 17, the Göttingen mini pig was used; in the other experiments the Munich mini pig.

5. Catheter technology used

There were the Infiltrator ^® catheter of _VT (San Diego, CA, USA), model DD 140015 (see DE 197 29 769) and the Victor ^® -i stent from Medtronic (Minneapolis, MN, USA), Model 6320 uses.

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

6.1 Production of the transfection medium

In the case of solution 15, a solution of 250mM sucrose and 25mM 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. 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.

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 in half the required

Pipette the 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 emerging

Solution was mixed by pulling it into the pipette tip several times or by rotating the closed reaction vessel.

As a typical example, the following preparation of a DAC-30 / DNA mixture with a lipid-DNA ratio of 4: 1 (w / w) is intended 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 obtained by adding

Transfection solution brought to a final volume of 750 ul and through

Mix homogenized with the vortex shaker. Four times the amount of DAC-30, i.e. 30 μg (at a concentration of 2 μg / μl accordingly

15 μl), was also taken up in 750 μl transfection solution and homogenized by mixing with the vortex shaker. Then 750 ul

Pipette the DAC solution slowly dropwise into the 750 μl of the DNA solution.

The resulting solution contained 1.5 ml total volume of lipid-DNA complexes with a DNA concentration of 5 μg / ml. That solution was Stable over a period of at least 48 h at 4 ° C. and could either be used immediately or lyophilized as described below and reconstituted in 1.5 ml of water. 400 μl of this solution could then be injected into the porcine femoral artery as described below, which corresponds to a therapeutic dose of 2 μg DNA.

6.4 Scheme and implementation of 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 hours 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 of sterile, pyrogen-free water immediately before the operation. The redissolution was checked optically.

6.7 Transfection of the Vascular Wall Using an Infiltrator Catheter In Vivo (Mini Pig Model) A mini pig was intubated, anesthetized and used for surgical purposes

Intervention prepared, followed by a presentation 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. From this The prospective transfection sites were determined using a still image. The next step was to position the guidewire in the left femoral artery and to display the angiography of the correct position of the guidewire, as well as to open and position the IVT injection catheter 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 20s 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 at

Plastic preparations 4 μm 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 confirmed by the detection of

Nitrite was detected in the cell culture supernatant as follows: 100 μl of the sample to be examined were placed in a flat-bottom plate with 96 wells

Pipetted cell culture supernatant. 50 μl of each were added to the samples 2% sulfanilamide solution in 2.5% H ₃ PO ₄ and 50 μl of a 0.2% naphthylethylenediamine solution in 2.5% H ₃ PO were added. After 5 min incubation at room temperature, the absorption of the samples at 540 nm was determined 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 nitrogen oxide synthase in vitro: DAC-30, DMRIE-C and FuGENE ™ 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 KC1, 10 mM Tris / Cl pH 7.6). Smooth muscle cells from the pig aorta were cultivated in cell culture dishes with 6 wells each. In each case 500 μl of transfection solution (containing DNA in a concentration of 16 μg / ml) were added to the cells, incubated for 1 h at 37 ° C. and, after the solutions had been replaced by 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 1.7).

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 nitrogen oxide synthase in vivo: DAC-30, DMRIE-C and FuGENE ™

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 of 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 ™ was used 1: 1 (w / v) mixed in BSS buffer (137 mM NaCl, 5.4 mM KC1, 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 ™ / iNOS-DNA or DMRIE-C / iNOS-DNA. In view of the vztro 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 pig's aorta (see Example 1) were cultivated in cell culture dishes with 6 wells each and with liposome DNA

Complexes consisting of DAC-30 and the plasmid pAH 1 (human iNOS cDNA) transfected. To prepare the transfection solution (0.9% NaCl, 2 mM CaCl ₂ ) DAC-30 was mixed with 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. 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) or to untransfected cells (medium) (see 1.7). 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 nitrogen oxide synthase by liposomal complexes containing DAC-30 / iNOS-DNA was dependent on the lipid-DNA ratio in the preparation of the complexes. The expression of the transgene was optimal for a lipid-DNA ratio of 5: 1 at a constant dose of 4 μg iNOS plasmid (concentration 8 μg / ml). At the same time, these liposomal complexes showed low cytotoxicity when transfected in vitro. With a suitable liposome-DNA ratio of 5: 1, liposome-DNA complexes containing DAC-30 / iNOS-DNA showed an optimal expression efficiency at a DNA concentration of 4 μg / ml to 8 μg / ml and a 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 (0.9% NaCl 2 mM CaCl ₂ ). For the production of liposome DAC-30 DNA complexes were used in a weight ratio of 2: 1, 3: 1, 4: 1, 5: 1, 6: 1 and 8: 1 for 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 large, depending on the amount of DNA, affects 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 prepare 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 ™ 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 iNOS plasmid at a dose of 2 μg (corresponding to a concentration of 6.7 μg / ml) complexes of a smaller particle size (approx. 580 nm) than at a dose of 10 μg iNOS plasmid (corresponding to a concentration of 33.3 μg / ml (approx. 1000 nm When transfected into the vascular wall of the pig arteria in vivo, these mixtures showed a 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 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 pAHl (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. The induction was then carried out

Neointima formation a Wiktor-l stent (Medtronic) placed at each transfection site. After a test period of 6 weeks, the transfected vessel sections were removed. The evaluation of thin sections of the removed vessels was carried out quantitatively by Moφhometrie in comparison to the control (plasmid pcDNA3) (n = 3).

Result: The expression of the iNOS after transfection of solutions containing DAC-30 / iNOS DNA into the femoral artery of the pig in vivo caused a reduction in neointima formation (determined as the ratio of neointima to media) by approximately 20% (data not shown). 7. Time-dependent stability of lipid-DNA complexes

Experiment: Lipid-DNA mixtures were prepared consisting of DAC-30 and the plasmid pAH 9 (human iNOS cDNA) in a ratio of 4: 1 and 5: 1 with an increasing DNA concentration of 2.3 μg / ml, 6 , 7 μg / ml and 13.3 μg / ml in solution 1 1 (0.9% NaCl). The size of the resulting liposome-DNA complexes was determined immediately after production or after a standing time of 3 h at 4 ° C. by photon correlation spectroscopy (Zetasizer).

Result: Lipid-DNA mixtures containing DAC-30 / iNOS-DNA with a lipid-DNA ratio of 4: 1 formed complexes in a defined size range from approx. 540 to 700 nm, which even with increasing DNA concentration of 2 , 3 μg / ml, 6.7 μg / ml and 13.3 μg / ml remained constant at 4 ° C. over a period of 3 h. This constant particle size is a measure of the stability of the complexes. In contrast, with a lipid-DNA ratio of 5: 1, the size of the complexes increased 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 Room temperature as follows: at 2.3 μg / ml 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. This 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 was prepared consisting of DAC-30 and the plasmid pAH 9 (human iNOS cDNA) in a ratio of 4: 1 at a constant DNA concentration of 5 μg / ml. These liposome-DNA complexes were prepared in various 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 was determined either immediately after production or after storage at 4 ° C. for 3 h, 24 h or 48 h by photon correlation spectroscopy (Zetasizer).

Result: Lipid-DNA mixtures containing DAC-30 / iNOS-DNA with a lipid-DNA ratio of 4: 1 and a DNA concentration of 5 μg / ml formed complexes in a defined size range from approx. 460 to 610 nm , This complex size remained largely constant in the transfection solutions L 13, L 14 and L 15 over a period of 3 h to 48 h at 4 ° C (increase approx. By factor 1.2 in solution 13, approx. By factor 1.4 in Solution 14 or about 1.1 in solution 15), ie the complexes formed were stable. DAC-3 O / DNA complexes, on the other hand, which had been prepared in L 11, showed a significant increase in particle size by a factor of 2.5, i.e., over a period of 3 h to 48 h. the complexes formed were unstable. A slight increase in particle size, approximately by 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 ll (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 1.7). 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 1 1 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 pAHl (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 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 vessel 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-3 O / DNA complexes in solution 15

Experiment: A lipid-DNA mixture was prepared consisting of DAC-30 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 determined immediately after preparation or reconstitution of the lyophilized complexes (0 h) and after a standing time of 3 h at 4 ° C. by 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 over a period of 3 h a constant particle size (size increase less than 15%). This constant particle size over a period of at least 3 hours advantageously remains stable even after lyophilization of the complexes and subsequent reconstitution in sterile water. Lyophilization therefore has no influence on the short-term stability of the liposome-DNA complexes. (Data not shown).

12. Long-term stability of DAC-3 O / DNA complexes in solution 15

Experiment: A lipid-DNA mixture was prepared consisting of DAC-30 and the plasmid pAH 9 (human iNOS cDNA) in a ratio of 4: 1 in solution 15 (250 mM sucrose, 25 mM NaCl). The DNA concentration was 2.5 μg / ml, 5 μg / ml and 10 μg / ml, respectively. The particle size of the complexes produced was determined immediately (0 h) or after a standing time of 3 h, 24 h and 48 h by photon correlation spectroscopy (Zetasizer). The transfection solutions prepared in this way contained a total dose of 1 μg, 2 μg or 4 μg plasmid DNA and were transfected immediately (0 h) or after a standing time of 3 h, 24 h and 48 h in COS-7 cells. The accumulated nitrite was detected in the cell culture supernatant 48 h after transfection (see 1.7). 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 standing time of the transfection solution of up to 48 h. DAC-3 O / DNA complexes prepared in solution 15 advantageously showed a constant particle size and expression efficiency over a service life of the transfection solution of up to 48 h, which was retained even with increasing DNA concentration. (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 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. 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 nitric oxide synthase was evaluated quantitatively by Mohometry 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 transfection efficiency in vivo at a dose of 2 μg iNOS plasmid than 0.5 μg or 1 μg (approx. 43% compared to approx. 31.3% and 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 the transfection of one site of the right or left femoral artery of the pig in vivo by means of Infiltrator ^® catheter was used freshly prepared (dose 2 μg DNA) or lyophilized and reconstituted in sterile water (0.5; 1; and 2 μg DNA). 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 Moφhometrie.

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

15. Transfection efficiency in vivo with lyophilized / reconstituted complexes: DNA dose dependency

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 pAH9 (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 locations each on the right and left femoral arteries Pigs 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 each 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 pAH9 (human iNOS cDNA) or the control plasmid pAH 7 (without cDNA insertion) in a ratio of 4: 1 in solution 15. The solution was lyophilized and Reconstituted in vivo in sterile water immediately before transfection into one site of the right and left femoral artery of the pig using Infiltrator ^® catheter. A Wiktor ^® -i stent (Medtronic) was then placed at each transfection site to induce neointima formation. After a test period of 28 days, the transfected vessel sections were removed. The evaluation was carried out by intravascular ultrasound examination of the transfected vascular sections 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 entire vessel area (n = 8).

Result: Liposomes / DNA complexes prepared in solution 15 containing a dose of 1 μg iNOS-DNA / DAC-30 after lyophilization and reconstitution in sterile water reduced the neointima formation in the pig restenosis model by approx. 42% in vivo , (Data not shown).

17. Effectiveness 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 plasmid pAH 7 (without cDNA insertion) in a ratio of 4 : 1 in solution 15. The solution is lyophilized and reconstituted in vivo in sterile water using an Infiltrator ^® catheter immediately before transfection into one site of the right and left femoral artery of the pig. A Wiktor ^® -i stent (Medtronic) is then placed at each transfection site to induce neointima formation. After a test period of 28 days, the transfected vessel sections are removed. The evaluation is carried out by intravascular ultrasound examination of the transfected vascular sections and evaluates the reduction in the neointima formed after treatment with iNOS plasmid compared to the control plasmid as a ratio of the plaque area to the entire vascular area.

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

Rabbits weighing 3 to 3.5 kg are used for the experiment to prevent graft arteriosclerosis. The animals are anesthetized for the surgical procedure, which is carried out under sterile conditions. The Gene transfer into the donor vessel is carried out by means of an Infiltrator ^® catheter, which is introduced into the iliac artery via a guide wire via the exposed right carotid artery through a sheath. The HO-1 expression plasmid is injected into the vascular wall of the iliac artery using an Infiltrator ^® catheter at low balloon pressure (approx. 0.6 atm.). 100-150 μl of transfection solution consisting of DAC-30 and an HO-1 expression plasmid in a ratio of 4: 1 in solution 15 are injected into the vessel wall within about 30 seconds. The seat of the inflated catheter is checked angiographically. Following the removal of the catheter, the transfected segment of the vessel is removed and inserted at the appropriate location in the iliac artery of an allogeneic recipient rabbit. After 2-4 months, histochemistry is used to investigate whether, compared to control-treated animals, the development of arteriosclerotic changes in the implanted vessels (intimal hypothyroidism, leukocyte infiltration) can be reduced.

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

Rabbits weighing 3-3.5 kg (New Zealand White rabbits) are used for the experiment to induce the growth of collateral vessels. The animals are anesthetized for the surgical procedure, which is carried out under sterile conditions. The exposed femoral artery is tied off by two ligatures at a distance of 1.5-2 cm so that the branches of the profunda femoris artery, the arteria circumflexa femoris lateralis and the arteria circumflexa abdominis remain continuous. An infiltrator catheter is used for the gene transfer, which is inserted into the femoral artery via a guide wire via the exposed right carotid artery through a sheath. The MCP-1 plasmid is injected into the vessel section proximal to the ligature using an Infιltrator ^® catheter at low balloon pressure (approx. 0.6 atm.). 100-150 μl Transfection solution consisting of DAC-30 and an MCP-1 expression plasmid in a ratio of 4: 1 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 7 days, angiography is used to determine whether more collaterals have formed compared to control-treated animals. Histological methods are used to investigate the proliferation of endothelial cells and smooth muscle cells in the vascular system of the treated tissues compared to control treated animals.

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

Rabbits weighing 3-3.5 kg (New Zealand White rabbits) are used for the experiment to induce the growth of collateral vessels. The animals are anesthetized for the surgical procedure, which is carried out under sterile conditions. The exposed femoral artery is tied off by two ligatures at a distance of 1.5-2 cm so that the branches of the profunda femoris artery, the arteria circumflexa femoris lateralis and the arteria circumflexa abdominis remain continuous. Approximately 7-21 days after vascular occlusion, a gene transfer is carried out with plasmid DNA, which leads to the expression of MCP-1 or GM-CSF in the vascular wall. An infiltrator catheter is used for the gene transfer, which is inserted into the femoral artery via a guide wire via the exposed right carotid artery through a sheath. The plasmid DNA is injected into the vessel section proximal to the ligature using an Infiltrator ^® catheter at low balloon pressure (approx. 0.6 atm.). 100-150 μl of transfection solution consisting of DAC-30 and plasmid DNA in a ratio of 4: 1 in solution 15 are injected into the vessel wall within about 30 seconds. 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 more collaterals have formed compared to control treated animals. Histological methods are used to investigate the proliferation of endothelial cells and smooth muscle cells in the vascular system of the treated tissues compared to control treated animals.

Claims

claims

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 carbamoylene 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 about 300-800 nm.

2. Pharmaceutical composition according to claim 1, wherein the size of the nucleic acid-lipid complexes is in a range of about 350-550 nm.

3. Pharmaceutical composition according to claim 1 or 2, wherein the nucleic acid (N) codes for one of the isoforms of nitrogen oxide synthase (NOS), hemoxygenase (HO), monocyte chemoattractant protein (MCP), or a variant of one of these proteins.

4. Pharmaceutical composition according to one of claims 1-3, wherein 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 encoded for the respective human form.

5. Pharmaceutical composition according to one of claims 1-4, wherein the cationic lipid (KL) 3ß- [N- (N, N'-dimethylaminoethane) - carbamoyljcholesterol (DAC-Chol) or 3ß- [N- (N ', N '-Dimethylamino-ethane) -carbamoyl] cholesterol (DC-Chol).

6. Pharmaceutical composition according to one of claims 1-5, wherein the non-cationic lipid (NKL) is a lipid selected from at least one phosphatidylcholine, at least one phosphatidylethanolamine and / or cholesterol.

7. Pharmaceutical composition according to claim 6, wherein the phosphatidylethanolamine is a diacylphosphatidylethanolamine with a chain length of 10-28 carbon atoms, preferably dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine

(DPPE) and / or dioleylphosphatidylethanolamine (DOPE).

8. The pharmaceutical composition according to claim 7, wherein the cationic lipid (KL) is DAC-Chol and the non-cationic lipid (NKL) is DOPE, 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. Pharmaceutical composition according to one of claims 1-8, wherein 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 approx. 5: 1, in each case based on the weight.

10. Pharmaceutical composition according to one of claims 1-9, wherein the composition is present as a solution or as a lyophilisate.

1 1. Pharmaceutical composition according to any one of claims 1-10, wherein said excipient is a stabilizing agent, in particular at least one sugar, at least one polyhydric alcohol and / or at least one inorganic salt.

12. A pharmaceutical composition according to any one of claims 1-11, wherein said additive is at least one molecule specifically recognizing the target cells and / or at least one molecule facilitating gene transfer into the cells.

13. A method for producing the pharmaceutical composition according to any one of claims 1-12, characterized by the following steps: (i) providing a mixture of a cationic lipid (KL) according to 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; (ii) mixing the mixture of (KL) and (NKL) with the nucleic acid (N); (iii) optionally lyophilizing; and (iv) optionally reconstitute.

14. The method according to claim 13, wherein in step (ii) the total lipid from (KL) and (NKL) and the nucleic acid (N) in a ratio of about 1: 1 to about 10: 1, preferably about 4: 1 or approx. 5: 1, each based on the weight.

15. The method according to claim 13 or 14, wherein in step (i) the provision of the mixture of (KL) and (NKL) and / or the provision of the nucleic acid (N) using a stabilizing agent, in particular at least one sugar, at least one polyhydric alcohol and / or at least one inorganic salt.

16. The method of claim 15, wherein the stabilizing agent is used in the form of an isoosmotic aqueous solution.

17. Pharmaceutical composition obtainable by the method according to any one of claims 13-16.

18. Use of the pharmaceutical composition according to any one of claims 1-12 or 17 in the manufacture of a medicament for use in gene therapy including a combination therapy with pharmacological agents.

19. Use according to claim 18 for the treatment of vascular diseases, genetically caused diseases and or diseases which can be treated by gene transfer, including their prevention.

20. Use according to claim 18 or 19 for the treatment and prevention of peripheral and / or coronary vascular diseases.

21. Use according to claim 20, wherein the vascular disease is the stenosis of vessels, including vascular grafts, the restenosis after a ner percutaneous transluminal angioplasty (PTA) of coronary and / or peripheral vessels, a disease due to insufficient blood flow to tissues, coronary artery disease, the myocardial infarction, vascular arteriosclerosis and or a disease which is used to reject vascular and / or Organ transplants is.

22. Use according to any one of claims 18-21 for local somatic gene therapy.

23. Use according to claim 22, wherein the gene therapy is carried out using a catheter, in particular an infiltrator catheter.

24. Use according to claim 22 or 23, wherein the pharmaceutical composition with a total dose in a range of about 0.1 - 20 ug, preferably about 0.5 - 10 ug, particularly preferably about 1 - 5 ug, each based on the total amount of nucleic acid per application.

25. Use of an 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.

26. Use of an 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 during lyophilization and / or reconstitution.

27. Use according to claim 25 or 26, wherein the disaccharide is sucrose and the inorganic salt is sodium chloride.