EP1216027A2 - Composition pharmaceutique sous forme d'un complexe acide nucleique-lipide, sa preparation et son utilisation en therapie genique - Google Patents

Composition pharmaceutique sous forme d'un complexe acide nucleique-lipide, sa preparation et son utilisation en therapie genique

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Publication number
EP1216027A2
EP1216027A2 EP00967678A EP00967678A EP1216027A2 EP 1216027 A2 EP1216027 A2 EP 1216027A2 EP 00967678 A EP00967678 A EP 00967678A EP 00967678 A EP00967678 A EP 00967678A EP 1216027 A2 EP1216027 A2 EP 1216027A2
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Prior art keywords
nucleic acid
pharmaceutical composition
lipid
dna
dac
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EP00967678A
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German (de)
English (en)
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Jens Schletter
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Cardion AG
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Cardion AG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Definitions

  • 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
  • compositions in the form of a nucleic acid-lipid complex 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.
  • DOPE dioleylphosphatidylethanolamine
  • 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.
  • 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 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
  • 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).
  • vascular diseases it is preferably one with vasodilator and or vascular properties.
  • 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).
  • HO-1 hemoxygenase 1
  • HO-2 hemoxygenase 2
  • HO-3 hemoxygenase 3
  • 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 ,
  • 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).
  • CO free iron and carbon monoxide
  • the latter like the nitrogen oxide (NO) generated by NOS, exerts a vasodilator and antithrombotic effect.
  • HO-1 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.
  • the present invention also includes variants of the aforementioned proteins.
  • 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.
  • nucleic acids from different human cells or tissues or allelic variants 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.
  • 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.
  • 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.
  • 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
  • DOPE Dioleylphosphatidylethanolamine
  • 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.
  • the ratio of the cationic lipid to the non-cationic lipid can also be expressed as a molar ratio.
  • 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.
  • 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.
  • 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.
  • a sugar is also understood to mean a sugar alcohol such as mannitol.
  • An example of a polyhydric alcohol is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the combination of sucrose as Sugar and NaCl as the inorganic salt are particularly preferred.
  • 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.
  • 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".
  • 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.
  • 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).
  • a number of other molecules are conceivable, which can also contribute to the specific recognition of a target cell.
  • 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.
  • nucleic acid molecules 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:
  • 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.
  • 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.
  • 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.
  • a stabilizing agent in particular at least one sugar, at least one inorganic salt and / or at least one polyhydric alcohol.
  • 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.
  • 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.
  • 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).
  • 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.
  • Another preferred embodiment is the use of the pharmaceutical composition described herein for local somatic gene therapy.
  • 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).
  • a minimally invasive and efficient catheter technology with which individual vessel sections can be transfected in vivo.
  • 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 ⁇ g (including all values in between), based on the total amount of nucleic acid.
  • 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
  • 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 ⁇ g to approximately 10 ⁇ g, particularly preferably approximately 1 ⁇ g to approximately 5 ⁇ g, in each case based on the total amount of nucleic acid.
  • iNOS in addition to a vasodilator and antithrombotic effect, an antiproliferative effect on the smooth muscle cells of the vascular wall can be achieved.
  • a better transfection efficiency was surprisingly found in vivo with a total dose of 2 ⁇ g DNA than with a total dose of 10 ⁇ g.
  • HO-1 is used as a therapeutically active gene, a vasodilator and antithrombotic effect can be achieved.
  • HO-1 is described in transplant models as anti-inflammatory and immunoprotective (Hancock et al. (1998), supra; and Soares et al. (1998), supra).
  • 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.
  • 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.
  • 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.
  • arteriogenesis ie the formation of collateral vessels from existing arterioles
  • angiogenesis ie the formation of new capillaries
  • 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.
  • CSFs Coldy-stimulating factors
  • M-CSF also called CSF-1, as well as its alternative splice forms such as CSF-4
  • CSF-1 has a specific effect on macrophages
  • G-CSF CSF-3
  • GM-CSF CSF-2
  • 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).
  • 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.
  • a further aspect of the invention relates to the use of an isoosmotic composition
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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
  • 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 Inf ⁇ ltrator ® catheter and implantation of a Wiktor ® -i- stents)
  • DAC-30 which is available from G.O.T. (Berlin) is distributed.
  • DAC-30 TM is the trade name for a mixture of DAC cholesterol and the neutral lipid DOPE im
  • DAC-Chol DOPE
  • DAC-40 40:60
  • DAC-50 50:50
  • DMRIE-C 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammonium bromide
  • the plasmid pSCMV-iNOS contains the cDNA sequence of the murine iNOS. Its manufacture is described in DE 44 11 402.
  • 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.
  • 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-
  • 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.
  • Solution was mixed by pulling it into the pipette tip several times or by rotating the closed reaction vessel.
  • 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 ⁇ g: 7.5 ⁇ l DNA (at a
  • the resulting solution contained 1.5 ml total volume of lipid-DNA complexes with a DNA concentration of 5 ⁇ g / 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 ⁇ l of this solution could then be injected into the porcine femoral artery as described below, which corresponds to a therapeutic dose of 2 ⁇ g DNA.
  • 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:
  • the temperature of the floor space has risen by 10 ° C / h to 20 ° C.
  • the samples were taken under sterile conditions.
  • the lyophilisates were stored at 4 ° C until used.
  • 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.
  • Injection catheter at 3.0 bar. Angiography was then performed to check for complete vascular occlusion. A manual injection of 300 ⁇ l or 400 ⁇ l 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 ⁇ m and 10 ⁇ m for frozen sections were 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.
  • Nitrite was detected in the cell culture supernatant as follows: 100 ⁇ l of the sample to be examined were placed in a flat-bottom plate with 96 wells
  • Smooth muscle cells from the pig aorta were cultivated in cell culture dishes with 6 wells each.
  • 500 ⁇ l of transfection solution (containing DNA in a concentration of 16 ⁇ g / 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).
  • 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).
  • Transfection solutions were prepared consisting of cationic liposomes and the plasmid pSCMV-iNOS (murine iNOS-cDNA).
  • a dose of 10 ⁇ g 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 TM was used 1: 1 (w / v) mixed in BSS buffer (137 mM NaCl, 5.4 mM KC1, 10 mM Tris / Cl pH 7.6).
  • DAC-30 Expression and toxicity in vitro depending on the lipid-DNA ratio
  • DAC-30 Complexes consisting of DAC-30 and the plasmid pAH 1 (human iNOS cDNA) transfected.
  • DAC-30 was mixed with a constant dose of 4 ⁇ g DNA (corresponding to a concentration of 8 ⁇ g / ml) in a ratio of 2: 1, 4: 1, 5: 1, 6: 1 and 8: 1.
  • DAC-30 was mixed with a constant dose of 4 ⁇ g DNA (corresponding to a concentration of 8 ⁇ g / ml) in a ratio of 2: 1, 4: 1, 5: 1, 6: 1 and 8: 1.
  • DAC-30 was mixed with a constant dose of 4 ⁇ g DNA (corresponding to a concentration of 8 ⁇ g / ml) in a ratio of 2: 1, 4: 1, 5: 1, 6: 1 and 8: 1.
  • a DNA dose of 2 ⁇ g / ml, 4 ⁇ g / ml, 8 ⁇ g / ml or 16 ⁇ g / ml was used to prepare
  • transfection solution 500 ⁇ l 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).
  • 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 ⁇ g iNOS plasmid (concentration 8 ⁇ g / ml). At the same time, these liposomal complexes showed low cytotoxicity when transfected in vitro.
  • liposome-DNA complexes containing DAC-30 / iNOS-DNA showed an optimal expression efficiency at a DNA concentration of 4 ⁇ g / ml to 8 ⁇ g / ml and a low cytotoxicity up to a DNA concentration of 8 ⁇ g / ml. (Data not shown).
  • a transfection solution was prepared consisting of complexes of DAC-30 and the plasmid pAH 9 (human iNOS cDNA) in
  • Lipid-DNA mixtures containing DAC-30 / iNOS-DNA formed at a constant DNA concentration of 6.7 ⁇ g / 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).
  • 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 ) .
  • DAC-30 was used in a ratio of 5: 1 to the plasmid DNA at a DNA dose of 2 ⁇ g (concentration 6.7 ⁇ g / ml) or 10 ⁇ g DNA (concentration 33.3 ⁇ g / ml).
  • the size of the complexes formed was determined by photon correlation spectroscopy (Zetasizer).
  • 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.
  • 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 ⁇ g (corresponding to a concentration of 6.7 ⁇ g / ml) complexes of a smaller particle size (approx. 580 nm) than at a dose of 10 ⁇ g iNOS plasmid (corresponding to a concentration of 33.3 ⁇ g / ml (approx.
  • Transfection solutions were prepared consisting of DAC-30 and 0.7 ⁇ g (corresponding to a DNA concentration of 2.3 ⁇ g / ml) of the plasmid pAHl (human iNOS cDNA) or the control plasmid pcDNA3 (without cDNA-
  • 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 ⁇ g / ml, 6 , 7 ⁇ g / ml and 13.3 ⁇ g / 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).
  • 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 ⁇ g / ml, 6.7 ⁇ g / ml and 13.3 ⁇ g / 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.
  • the size of the complexes increased with an increasing amount of DNA of 2.3 ⁇ g / ml, 6.7 ⁇ g / ml and 13.3 ⁇ g / ml over a period of 3 h Room temperature as follows: at 2.3 ⁇ g / ml by a factor of 2 from approx. 650 nm to approx. 1280 nm, at 6.7 ⁇ g / ml by a factor of 3 from approx. 740 nm to approx. 2200 nm and at 13.3 ⁇ g 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).
  • 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 ⁇ g / 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).
  • Lipid-DNA mixtures containing DAC-30 / iNOS-DNA with a lipid-DNA ratio of 4: 1 and a DNA concentration of 5 ⁇ g / 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)
  • 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).
  • DAC-30 was added in a ratio of 4: 1 to a constant amount of DNA of 2 ⁇ g (corresponding to a concentration of 5 ⁇ g / ml).
  • the liposome-DNA complexes were transfected into COS-7 cells immediately after production.
  • Transfection solutions were prepared consisting of DAC-30 and 2 ⁇ g 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 ).
  • Liposome-DNA complexes containing DAC-30 / iNOS-DNA in L 15 showed a better transfection efficiency in vivo at a dose of 2 ⁇ g 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).
  • 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
  • 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).
  • Lipid-DNA mixtures containing DAC-30 / iNOS-DNA showed a lipid-DNA ratio of 4: 1 and an increasing DNA concentration of 2.5 ⁇ g / ml, 10 ⁇ g / ml, 25 ⁇ g / ml and 50 ⁇ g / 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).
  • 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 ⁇ g / ml, 5 ⁇ g / ml and 10 ⁇ g / 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).
  • 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 ⁇ g / ml, 5 ⁇ g / ml, and 10 ⁇ g / 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).
  • Transfection solutions were prepared consisting of DAC-30 and 0.5 ⁇ g, 1 ⁇ g or 2 ⁇ g 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 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 nitric oxide synthase was evaluated quantitatively by Mohometry compared to the control (plasmid pAH 7 or untreated vessel).
  • Liposome-DNA complexes containing DAC-30 / iNOS-DNA in solution 15 showed a better transfection efficiency in vivo at a dose of 2 ⁇ g iNOS plasmid than 0.5 ⁇ g or 1 ⁇ g (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
  • 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.
  • pAH 9 human iNOS cDNA
  • 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 Mo ⁇ hometrie.
  • a transfection solution containing DAC-30 / iNOS-DNA in solution 15 (dose of 2 ⁇ g 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 ⁇ g); control (2 ⁇ g); reconstituted transfection solution (0.5; l; and 2 ⁇ g DNA)).
  • Transfection solutions were prepared consisting of DAC-30 and 0.7 ⁇ g, 1 ⁇ g, 1.5 ⁇ g, 2 ⁇ g and 4 ⁇ g 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 TM 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 ⁇ m sections at intervals of 1 mm each over the entire transfected vascular section.
  • Liposome-DNA complexes containing iNOS-DNA / DAC-30 as reconstituted lyophilisate in a dose of 2 ⁇ g plasmid DNA showed better transfection efficiency in vivo than in a dose of 0.7 ⁇ g, 1 ⁇ g, 1.5 ⁇ g or 4 ⁇ g (Fig. 8A).
  • the better transfection efficiency at a dose of 2 ⁇ g in vivo was not influenced by the implantation of a stent (see FIG. 8B; implantation of a Wiktor®- i stent).
  • a transfection solution was prepared consisting of DAC-30 and 1 ⁇ g 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.
  • a transfection solution is prepared consisting of DAC-30 and 0.25 ⁇ g, 0.5 ⁇ g or 1 ⁇ g 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.
  • 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.).
  • 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.
  • the transfected segment of the vessel is removed and inserted at the appropriate location in the iliac artery of an allogeneic recipient rabbit.
  • 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.
  • Rabbits weighing 3-3.5 kg 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 Inf ⁇ ltrator ® catheter at low balloon pressure (approx. 0.6 atm.). 100-150 ⁇ l 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.
  • Rabbits weighing 3-3.5 kg 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 ⁇ l 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.

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Abstract

Composition pharmaceutique sous forme d'un complexe acide nucléique-lipide, qui contient au moins un lipide cationique, au moins un lipide non cationique, au moins un acide nucléique codant pour une protéine destinée au traitement de maladies des vaisseaux, en particulier une protéine à propriétés vasodilatatrices et/ou angiogènes, et éventuellement des substances auxiliaires et/ou des adjuvants appropriés. Le lipide cationique (KL) contient un groupe dérivé du cholestérol auquel est relié au moins un groupe amino cationique choisi parmi des groupes amino primaire, secondaire et tertiaire et/ou un sel d'ammonium quaternaire, par l'intermédiaire d'un groupe de liaison, choisi parmi des carboxamides et des carbamoyles, et d'un bras d'espacement constitué par un groupe alkyle linéaire ou ramifié ayant 1 à 20 atomes de carbone. La taille du complexe acide nucléique-lipide est de l'ordre d'env. 300 à 800 nm. La présente invention concerne également la préparation de ladite composition pharmaceutique et son utilisation en thérapie génique.
EP00967678A 1999-09-15 2000-09-14 Composition pharmaceutique sous forme d'un complexe acide nucleique-lipide, sa preparation et son utilisation en therapie genique Withdrawn EP1216027A2 (fr)

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DE1999144262 DE19944262A1 (de) 1999-09-15 1999-09-15 Pharmazeutische Zusammensetzung in Form eines Nukleinsäure-Lipid-Komplexes, ihre Herstellung und Verwendung in der Gentherapie
PCT/EP2000/008996 WO2001019400A2 (fr) 1999-09-15 2000-09-14 Composition pharmaceutique sous forme d'un complexe acide nucleique-lipide, sa preparation et son utilisation en therapie genique

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US5658565A (en) * 1994-06-24 1997-08-19 University Of Pittsburgh Of The Commonwealth System Of Higher Education Inducible nitric oxide synthase gene for treatment of disease
DE4411402A1 (de) * 1994-03-31 1995-10-05 Juergen Schrader DNA-Expressionsvektoren zur Verwendung in der gentherapeutischen Behandlung von Gefäßerkrankungen
US6071890A (en) * 1994-12-09 2000-06-06 Genzyme Corporation Organ-specific targeting of cationic amphiphile/DNA complexes for gene therapy
US5888821A (en) * 1994-12-28 1999-03-30 Max-Delbruck-Centrum Fur Molekulare Medizin Cholesterol derivative for liposomal gene transfer
US5660855A (en) * 1995-02-10 1997-08-26 California Institute Of Technology Lipid constructs for targeting to vascular smooth muscle tissue
WO1996027393A1 (fr) * 1995-03-07 1996-09-12 University Of Pittsburgh Formulation de poudre seche pour therapie genique
EP0826063A1 (fr) * 1995-04-25 1998-03-04 Vical Incorporated Formulations en ampoule unidose de complexes adn/lipides
US5753262A (en) * 1995-06-07 1998-05-19 Aronex Pharmaceuticals, Inc. Cationic lipid acid salt of 3beta N- (N', N'-dimethylaminoethane) - carbamoyl!cholestrol and halogenated solvent-free preliposomal lyophilate thereof
WO1996040265A1 (fr) * 1995-06-07 1996-12-19 The Regents Of The University Of California Stabilisation de complexes de polynucleotides
EP0832271B8 (fr) * 1995-06-07 2005-03-02 INEX Pharmaceuticals Corp. Particules d'acides nucleiques et de lipides preparees au moyen d'un intermediaire de complexe hydrophobe d'acides nucleiques et de lipides et utilisation pour transferer des genes
US5811406A (en) * 1995-06-07 1998-09-22 Regents Of The University Of California Dry powder formulations of polynucleotide complexes
EP0941066B1 (fr) * 1996-08-26 2003-10-29 Transgene S.A. Complexes acides nucleiques/lipides cationiques
WO1998044934A1 (fr) * 1997-04-07 1998-10-15 Rutgers, The State University Of New Jersey Therapie genique de carcinomes fondee sur la cytokine et le cisplatine
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