WO1999008997A1 - Neue lipopolyamine, deren darstellung und anwendung - Google Patents
Neue lipopolyamine, deren darstellung und anwendung Download PDFInfo
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- WO1999008997A1 WO1999008997A1 PCT/EP1998/005156 EP9805156W WO9908997A1 WO 1999008997 A1 WO1999008997 A1 WO 1999008997A1 EP 9805156 W EP9805156 W EP 9805156W WO 9908997 A1 WO9908997 A1 WO 9908997A1
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- octadecyl
- hexadecenyl
- tetradecenyl
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- 0 C[N+](C(CCC*CCC*)*(CCC*)=O)[O-] Chemical compound C[N+](C(CCC*CCC*)*(CCC*)=O)[O-] 0.000 description 7
- IVOGTYHOZFVQRR-UHFFFAOYSA-N CCCCCCCCCCCCCCCCCCN(CCCCCCCCCCCCCCCCCC)CCN(CCCNC(OC(C)(C)C)=O)CCCNC(OC(C)(C)C)=O Chemical compound CCCCCCCCCCCCCCCCCCN(CCCCCCCCCCCCCCCCCC)CCN(CCCNC(OC(C)(C)C)=O)CCCNC(OC(C)(C)C)=O IVOGTYHOZFVQRR-UHFFFAOYSA-N 0.000 description 1
- BDLZRCWUKCPQCH-UHFFFAOYSA-N CN(CCCN(CCCN)CCCN)CCCN(CCCN)CCCN Chemical compound CN(CCCN(CCCN)CCCN)CCCN(CCCN)CCCN BDLZRCWUKCPQCH-UHFFFAOYSA-N 0.000 description 1
- KMBPCQSCMCEPMU-UHFFFAOYSA-N CN(CCCN)CCCN Chemical compound CN(CCCN)CCCN KMBPCQSCMCEPMU-UHFFFAOYSA-N 0.000 description 1
- HPZOQQHJSJGNCE-UHFFFAOYSA-N CN(CCCN)CCCN(CCCN)CCCN Chemical compound CN(CCCN)CCCN(CCCN)CCCN HPZOQQHJSJGNCE-UHFFFAOYSA-N 0.000 description 1
- VZWKEWMJFMHCPN-UHFFFAOYSA-N CN(CCCN)CCCNCCCN Chemical compound CN(CCCN)CCCNCCCN VZWKEWMJFMHCPN-UHFFFAOYSA-N 0.000 description 1
- RNOWLZYGCXSADG-UHFFFAOYSA-N CN(CCCNCCCN)CCCNCCCN Chemical compound CN(CCCNCCCN)CCCNCCCN RNOWLZYGCXSADG-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C211/00—Compounds containing amino groups bound to a carbon skeleton
- C07C211/01—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms
- C07C211/02—Compounds containing amino groups bound to a carbon skeleton having amino groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
- C07C211/14—Amines containing amino groups bound to at least two aminoalkyl groups, e.g. diethylenetriamines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
- C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C217/42—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having etherified hydroxy groups and at least two amino groups bound to the carbon skeleton
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
- C07C229/16—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
- C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
- C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
- C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S977/00—Nanotechnology
- Y10S977/902—Specified use of nanostructure
- Y10S977/904—Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
- Y10S977/915—Therapeutic or pharmaceutical composition
Definitions
- Positively charged lipids are in the form of liposomes, micelles or as such for the introduction of biologically active substances such as peptides, peptoids, proteins, PNA, antiviral active ingredients, but especially DNA, RNA , Antisense DNA / RNA or ribozymes in eukariotic cells (for example mammalian, plant or insect cells).
- Lipopolyamines are a special class of cationic lipids that show comparatively excellent transfection properties. Transfection is the introduction of genetic material into eukaryotic cells.
- DNA for example plasmids, cosmids, single-stranded or double-stranded
- RNA or related classes of substances such as antisense-DNA / RNA or ribozymes
- nucleic acids for the introduction of nucleic acids in eukaryotic and in particular in mammalian cells, a large number of methods, such as, for example, the CaPO 4 precipitation method, the DEAE dextran method, synthetic polyamines (polyethyleneimine, polylysine, PAMAM dendrimers) are methods which use receptor-mediated endocytosis use, electroporation, microbombardment, microinjection and methods using viral capsids as DNA carriers are known. Another method is called lipofection (PL Feigner et al., Proc. Natl. Acad. Sei. USA 74, 7413 1987).
- Liposomes are usually spherical arrangements of lipids in aqueous solutions with a "Büayer structure” and are typically divided into three classifications (see NY Academy Sciences Meeting: “Liposomes and their use in Biology and Medicine” by December over 1977): multilamellar vesicles (MLV, up to 10000 nm), small unilamellar vesicles (SUV,
- liposomes 20-50 nm and large unilamellar vesicles (LUV, 600-30000 ⁇ m).
- LUV large unilamellar vesicles
- a number of manufacturing methods for liposomes are known and are described in "Liposome Technology” (Gregoriadis, CFC Press, N.Y. 1984), in “Liposomes” (Ostro, Marcel Dekker, N.Y. 1987) or in review articles by Lichtenberg et al. (Methods Biochem. Anal. 33, 337-462, 1988), Pagano and Weinstein (Ann. Rev. Biophysic. Bioeng. 7, 435-68, 1978), or Szoka and Papahadjopoulos (Ann. Rev. Biophysic. Bioeng. 9 , 467-508, 1980).
- Known methods are, for example, the “reverse phase evaporation” method and the extrusion method, in which a lipid solution is pressed through a microporous membrane.
- Liposomes are typically also produced in the following way: The lipids are taken up in an organic solvent. Evaporation of the solvent under a stream of nitrogen creates a thin lipid ficus on the glass vessel wall. Adding water or aqueous buffer solution hydrates this film. The solution obtained is finally treated with ultrasound.
- Cationic lipids are becoming increasingly important in gene therapy.
- body cells are transfected using various methods by administering complexes of carrier and DNA intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, intranasally, in cerebrospinal fluid spaces or directly in tumors, or by removing, transfecting and reimplanting body cells.
- a method preferred in this context was the introduction of the genetic material by viral carriers.
- this method runs the risk of being mutated back to a pathogenic virus.
- the inserted DNA is stably incorporated into the genetic material, so that it is no longer possible to control the therapy or to return the cells to their original state.
- viral carriers have restrictions on the size of the DNA to be introduced. Modified DNA or RNA is not transmitted by viruses. In addition, only dividing cells can be transfected in this way.
- Transfection with cationic lipids is not subject to these restrictions.
- the transfection is usually transient, which means that the transfected DNA or RNA is only expressed for a certain time, since it is not incorporated into the genetic material and is degraded over time by nucleases. In this way, gene therapy can be dosed and made reversible.
- There are no restrictions on the size of the DNA and modified DNA or RNA eg antisense DNA / RNA or ribozymes stabilized by incorporation of modified nucleotides
- Non-dividing cells such as nerve cells, can also be transfected by cationic lipids.
- no immunogenic behavior of cationic lipids has been found in in vivo experiments.
- lipopolyamines There is a class of cationic lipids that take advantage of the known high affinity between polyamines (e.g. spermine, spermidin) and DNA for transfection, so-called lipopolyamines.
- the polyamines are linear or branched and contain ethylene; Propylene; or butylene groups between the amino functionalities.
- the polyamines are linked to a lipophilic residue in many different ways.
- the spermine positively charged at physiological pH is linked to a hydrophilic residue, in some cases via a spacer.
- Spermin forms stable complexes with DNA and similar compounds by being hydrogen bonded in the groove of the DNA.
- the first such lipospermine derivatives were from Behr, J. P. et al. (Proc. Natl. Acad. Sci. USA 86: 6982-6986; 1989; EP 0394111). They linked carboxyspermine to two different hydrophilic residues via a spacer.
- the structure of the 5-carboxyspenylglycine dioctadecylamide (DOGS) obtained is:
- DOGS is commercially available as Transfectam TM (Promega).
- DPES dipalmitoylphosphatidylethanolamine-5-carboxyspermylamide
- L-spermine-5-carboxyl-3- DL-1,2-dioleoyldimethylaminopropyl- ⁇ -hydroxyethylamine
- L-spermine-5-carboxyl-3- L-1,2-dipal_n toyl-dimethylam_nopropyl- ⁇ -hydroxyethylamine
- the object to be achieved according to the invention was therefore to find new cationic lipids with high effectiveness and the broadest possible spectrum of activity in combination with good stability and low toxicity.
- the Schenderend found that the combination of polyamines as a head group with a special lipophilic residue leads to particularly good properties in terms of efficiency and stability:
- the radicals R are saturated or unsaturated linear or branched alkyl chains, preferably having 10 to 20 carbon atoms.
- DNA-lipid complex has proven to be particularly important for successful transfection.
- DNA and cationic lipids come together, which are used as liposome or micelle formulations, there is a spontaneous condensation of the DNA with the breakdown of the hposomal or mice-like organizational structure of the cationic lipids.
- a DNA-lipid complex is formed, in which the DNA is embedded in a multilayer complex with a “structure” from said lipids (JO Rädler et al. Science, 275, 810, 1997).
- the sperm group As a DNA-affine head group, once bound to the DNA, the flexibility and symmetry of the rest of the molecular part (upophilic rest) are of particular importance, since the alkyl groups must be able to align themselves as much as possible in order to use Van der Waals Forces to be able to train stable "bilayer structure".
- the compounds proposed hitherto are generally not very symmetrical, which makes parallel alignment of the alkyl radicals more difficult, and include a total of esters, ethers or amide linkages between the alkyl radicals and a basic structure or a spacer. These compounds are therefore in their flexibility on account of the fixed bond angles and in the case of amide linkages (for example in the case of compounds from G. Byk et al, J. Med. Chem., 41. 224-235, 1998, EP 0394111, WO 9618372, WO 9746223 .WO 9802190 and WO 9802191) are severely restricted due to existing resonance structures.
- the O atom carries a negative charge and the amide N atom carries a positive charge.
- the result is a planar arrangement of the atoms involved in the amide bond. Free rotation of the amide bond between the carbonyl carbon and the nitrogen atom is not possible.
- This property has a very special effect on the structure of proteins, for example. (Secondary, tertiary and quaternary structure), whose peptide bonds are nothing more than amide linkages. Only the rigid, inflexible amide linkages give the protein its biologically necessary structure.
- the compounds according to the invention (see also below) link the alkyl radicals R 2 and R 3 via a tertiary nitrogen atom (amine).
- the present invention thus relates to new lipopolyamines of the general formula I which are able to transport biologically active molecules in eukaryotic cells:
- Ri is a residue of the following general formula:
- R 2 and R 3 independently of one another dodecyl, dodecenyl, tetradecyl, tetradecenyl, hexadecyl, hexadecenyl, octadecyl, octadecenyl or other alkyl radicals, which in all possible combinations may be saturated, unsaturated, branched, unbranched, fluorinated or non-fluorinated, and from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms are built up,
- g is> 1.
- compositions or formulations which contain liposomes or micelles which contain at least one of the compounds according to the invention with or without a co-epide, e.g. Dioleoylphosphatidylethanolamine (DOPE), Dioleoylphosphatidyl- choUn, Cholesterol or Cholesteryla in included.
- DOPE Dioleoylphosphatidylethanolamine
- Dioleoylphosphatidyl- choUn e.g. Dioleoylphosphatidylethanolamine (DOPE), Dioleoylphosphatidyl- choUn, Cholesterol or Cholesteryla
- DOPE Dioleoylphosphatidylethanolamine
- Cholesterol or Cholesteryla e.g. Dioleoylphosphatidyl- choUn
- Cholesterol or Cholesteryla e.g. Dioleoylphosphatidylethanolamine
- biologically active compounds such as DNA, RNA, ribozymes, antisense DNA, PNA, peptides, peptoids and proteins
- the compounds, compositions or formulations according to the invention having the (biologically active) to be introduced Complexes compounds and the complexes are brought into contact with eukaryotic cells in vivo or in vitro.
- compositions or formulations according to the invention for producing a medicament or a reagent
- biologically active compounds such as DNA, RNA, ribo enzymes, antisense DNA, PNA, peptides, peptoids and proteins in eukariotic cells in vivo or in vitro.
- compositions or formulations according to the invention for the manufacture of a medicament or a reagent
- biologically active compounds such as DNA, RNA, ribozymes, antisense DNA, PNA, peptides, peptoids and proteins
- Enhancers which enhance the action of the compounds according to the invention.
- the lipids according to the invention thus contain polyamines as DNA-affine head groups, which are possibly bound to a special upophilic residue via a spacer.
- a particular value of the lipids according to the invention lies in their stability in solution, with at the same time low toxicity for the cells in combination with exceptionally good transfection properties.
- the compounds according to the invention have better transfection properties when compared to Lipofectamin TM (Gibco-BRL: Life Technologies Inc.), the most effective transfection reagent from the group of lipopolyamines recognized to date in the art, if they have the wear the same head group.
- the transfection efficiency is not only higher in the serum-free and serum-containing milieu, but it also shows that a high level of transfection efficiency can be achieved by means of a much wider DNA-lipid ratio.
- the compounds according to the invention can be prepared by reacting an amino group of a diamine, if appropriate in the presence of a base, with an alkylating reagent and coupling the other amino group with a given, BOC-protected carboxy-polyalkylamen.
- the other amino group is preferably in the first reaction step e.g. protected with a BOC group, which is removed before reaction with the carboxypolyalkylamine.
- An alkyl bromide such as stearyl bromide can be used as the alkylation reagent and as the base e.g. Triethylamine or 4-methylmorpholine can be used.
- N- [2,5-bis [(3-aminopropyl) amino] -l-oxopentyl] - N ', N' -dioctadecylethylenediamine The starting point is alkylenediamine, in which an amino group is protected with N-tert-butyloxycarbonyl (BOC protected). The free amino group is derivatized by alkyl bromides such as stearyl bromide in the presence of a base. Then the protected
- the head group gives the desired compounds, depending on the work-up as a free amine or in the form of their salts.
- a diamine which is optionally protected on an amino group is reacted with acrylonitrile and the product is optionally reacted with an alkylating reagent, such as stearyl bromide, in the presence of a base.
- an alkylating reagent such as stearyl bromide
- a further preferred compound namely N, N-bis (3-aminopropyl) -N ', N ' - dioctadecylethylenediamine, can thus be prepared.
- This connection is based on N-tert.- Butyloxycarbonylethylenediamine (BOC-ethylenediamine) obtained by reacting with acrylonitrile, alky- ing with alkyl bromide and subsequent hydrogenation. The subsequent steps are used for purification.
- the polyamine head groups can be extended or branched polyamine head groups can also be generated. There is the possibility of generating the head group individually and then coupling to the upophuene rest to expand or extend already coupled head groups on the molecule.
- the following reaction scheme known per se which is also included in the previous example, can be used:
- reaction of ornithine with acrylonitrile should serve as an explicit example.
- the desired product can be obtained as the main amount via the amount of acrylonitrile and the correct choice of the reaction temperature.
- the hydrogenation of the nitriles to the amines and their protection by the BOC protective group are described in JP Behr et al. Proc. Natl. Acad. Sei., USA, 86, pp. 6982-6986, 1989 carried out analogously to the synthesis of tetra-BOC-carboxyspermine.
- a further possibility for the extension and branching of the polyamine head groups consists in the alkylation of amines with N- (tert-butyloxycarbonyl) -3-bromopropylamine:
- lipids listed can be obtained using the methods listed in the examples above and below (blocking and deblocking of amino functions with the BOC protecting group, alkylation of amino functions with alkyl bromides, formation of amide bonds using anologically known peptide chemistry, cyanoethylation of amino functions via acrylonitrile, hydrogenation of cyanofile to amines) or with the generally known methods familiar to organic chemists (alkylation of alcohols, formation of ester bonds, reduction of esters to ethers or amides to amines via lithium alanate).
- liposomes can also be formed with the above-mentioned lipids, or in combination with other lipids such as cholesterol, cholesterylamine, dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC).
- DOPE dioleoylphosphatidylethanolamine
- DOPC dioleoylphosphatidylcholine
- lysosomatotropic substances such as chloroquine or virus-derived lysosomatropic proteins.
- Enhancers enhance the effects of the cationic lipids.
- Enhancers that have no transfecting properties themselves, and those that can be used for transfection even without cationic lipids.
- the transfection efficiency of the catalytic lipids is simply amplified.
- the combined efficiency shows a transfection efficiency that is greater than that of the single application.
- the combination effect therefore comes about through a synergy effect.
- Enhancers usually work by precondensing the DNA to be transfected or they increase the permeability of the cell membrane to be penetrated. Examples of enhancers are polyethyleneimine, transferrin, protamine sulfate, polyhistones, fusogenic peptides, virus housings, viral surface peptides, replication-deficient viruses, etc.
- the lipopolyamines according to the invention are positively charged at physiological pH and can therefore bind stable aggregates with negatively charged macromolecules, in particular with DNA and related substance classes.
- the lipids according to the invention and their liposome formulations have no ZeU specificity.
- a targeted delivery by the lipids according to the invention or their liposome formulations can be achieved by balancing out the charges of lipids and the biomolecules to be transported and additionally adding internalization factors to the aggregate between the biomolecule and lipids.
- internation factors are transferrin, galactose, mannose, mannose-6-phosphate, asialglycoprotein, conalbumin, lectins, transcobalamin, ⁇ -2-macroglobulin , Organic tin, folate, mannose glycoproteins. Further are in EP 0535576, EP 0544292, WO
- zeU-specific antibodies can also be used.
- the compounds according to the invention can be used for therapeutic purposes.
- such compounds can be used for the gene therapy of, for example, cystic fibrosis, muscular dystrophy, phenylketonuria, maple syrup disease, propionic acidemia, methylmalonic acidemia, adenosine deaminase deficiency, hypercholesterolemia, hemophilia and ß-thalassemia.
- Gene therapy treatment methods are also of interest if hormones, growth factors, cytotoxins or immunomodulatory proteins are to be synthesized in the organism.
- DNA fragments can be brought into cells using these lipids, in which these DNA have the desired effect.
- the desired effect can be the replacement of missing or defective DNA regions or the inhibition of DNA regions (for example antisense DNA / RNA) which trigger the disease in the diseased ZeU type.
- tumor-suppressing genes can be used in cancer therapy or, through the introduction of cholesterol-regulating genes, can contribute to the prevention of heart and blood vessel diseases.
- DNA encoding ribozymes or ribozymes themselves can also be introduced into diseased cells.
- the translation of that DNA creates active ribozymes, which catalytically cleave m-RNA at specific sites and thus prevent transcription.
- viral m-RNA can be specifically cleaved without affecting another cellular m-RNA.
- the multiplication cycle of viruses e.g. HTV, herpes, hepatitis
- viruses e.g. HTV, herpes, hepatitis
- lipids can be found, for example, in vaccination methods which function in the body of humans and animals on the basis of the expression of DNA which encodes immunogenic peptides.
- lipid / DNA complexes are used as vaccines.
- the introduction of the DNA into the body cells leads to the expression of the immunogenic peptide and thus triggers the immune response.
- macromolecules such as PNA, peptides, peptoids or proteins
- they can be coated as such with the lipopolyamines according to the invention or in liposomes which contain as a component the lipopolyamines according to the invention, are enclosed or are adsorbed on their surface, if there is a negative net charge. If such aggregates are brought into contact with ZeUs, these molecules are transported through the ZeU wall.
- Therapeutic peptides have a beneficial effect on numerous diseases.
- Such peptides or proteins are, for example, lymphokines, interleukins, tumor necrosis factors or interferons, further growth factors, tissue plasminogen activator, factor VIII: c, granulocyte macrophage colony stimulating factor, erythropoietin, insulin, calcitonin, thymidine kinase and others.
- Toxic peptides such as ricin, diphtheria toxin and others can also be used therapeutically in a profitable manner.
- Peptoids can be successfully used as peptide analogues to prevent rapid enzymatic breakdown in the body.
- the lipids according to the invention are mainly used to complex negatively charged molecules because of their negative charge and to introduce them into cells.
- so-called "assembling systems" can also be used to transport positively charged molecules by first complexing negatively charged liposomes with this positively charged molecule. If the ratios are chosen so that a negative net charge remains, these complexes can be used with these lipopolyamines according to the invention as such or in the form of liposomes by bringing the oppositely charged components into contact and the resulting positively charged overall complexes are taken up by the cells.
- cationic lipids can be found in the publications WO 9011092, WO 9116024, WO 9303768, Science 258, 744-746, 1992, which are incorporated herein by reference.
- Dulbecco 's modified Eagels Medium (DMEM), Gibco-BRL: Life Technologies Inc.
- FCS Fetal calf serum
- N'.N'-bis (2-cyanoethyl) -N-tert-butyloxycarbonylethylene diamine 500 mg (3 12 mmol, MW 160.22) N-tert-butyloxycarbonylethylene diamine are dissolved in 5 ml of water and 0 65 ml (9 8 mmol , MG 53 06) Acrylonitrile added Then stirred for 7 days at room temperature Then coated with ethyl acetate, shaken out and the organic phase separated. After drying over sodium sulfate, rotated off
- the trifluoroacetate obtained is dissolved in 40 ml of dry acetonitrile together with 2 43 g (7 28 mmol, MW 333 4) and octadecyl bromide and 2 23 ml (1 68 g, 13 02 ml, MW 129 25, d 0 755) dissolved diisopropylethylamine (DIPEA) and boiled under reflux for 24 hours.
- DIPEA diisopropylethylamine
- N'.N'-bis (3-aminopropylV NN-dioctadecylethylenediamine (TFA salt) 200 mg (0.3 mmol, MW 665.12)
- N-tert.-butyloxycarbonyl-N ', N'-dioctadecylethylenediamine are over 2 ml of a Mixture of trifluoroacetic acid and methylene chloride (1: 3 v / v) dissolved. After two hours, evaporated dry and dried for 2 hours in a high vacuum.
- Example 4 Lip o somen / microforming
- Dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, cholesterol or cholesterylamine are dissolved in an organic solvent (e.g. chloroform).
- the lipid from Example 1 as such or in the form of its salt, for example trifluoroacetate salt, is dissolved in an organic solvent (e.g. chloroform).
- an organic solvent e.g. chloroform.
- the lipids as such and in the form of their TFA salts are dissolved in ethanol with or without co-peptides. Mixing with water gives solutions of different compositions.
- the cell lines CV-1, Heia S3 and NIH 3T3 are in "Dulbecco's-modified Eagle's medium” (DMEM), the 10% fetal calf serum (FBS), 2 mM L-glutamine (Gin), 0.1 mM non-essential amino acids (NEAA), 100 U / ml penicillin and 100 ⁇ g / ml streptomycin contains, cultivated in an incubator (5% CO atmosphere).
- DMEM Dulbecco's-modified Eagle's medium
- FBS fetal calf serum
- Gin 2 mM L-glutamine
- NEAA non-essential amino acids
- 100 U / ml penicillin and 100 ⁇ g / ml streptomycin contains, cultivated in an incubator (5% CO atmosphere).
- the ZeUen are plated on a 12-WeU micro titer plate with a density of 1-1.5 x 10 5 and incubated overnight to approximately 60-80% confluency.
- the cationic lipid e.g. 3,6,9,12 ⁇ l as an ethanolic solution or
- Liposome formulation was also dissolved in 50 ⁇ l serum and antibiotic-free DMEM.
- the two solutions are mixed in a polystyrene container and left to stand for 10-15 minutes in order to allow the lipid / DNA complex to build up.
- the cells are washed once with PBS (phosphate buffered saline).
- PBS phosphate buffered saline
- antibiotic-free DMEM with or without serum are added to the cells.
- the DNA / lipid complex was added to the cells and incubated for 6 hours in the incubator. Then it is added to 1 ml with serum and antibiotic-containing DMEM in such a way that the medium contains a final concentration of 10% serum.
- the ZeUen are cultivated for another 20 h. Then tested for ⁇ -galactosidase according to the manufacturer's instructions for the ⁇ -galactosidase assay kit. The developed O-nitrophenol was measured photometrically at 405 nm.
- N ' , N ' -Bis- (3-aminopropyl) - N, N-dioctadecylethylenediamine (TFA salt) was used as a liposome formulation with 50 mol% dioleoylphosphatidylethanolamine in aqueous sterile solution. Total amount of epid: 2 mg / ml.
- Lipofectamine TM (Gibco-BRL: Life Technologies Inc.) was purchased from the manufacturer.
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JP2000509683A JP4504559B2 (ja) | 1997-08-13 | 1998-08-13 | 新リポポリアミン、その調製および適用 |
AT98946333T ATE208369T1 (de) | 1997-08-13 | 1998-08-13 | Neue lipopolyamine, deren darstellung und anwendung |
US09/463,172 US6281371B1 (en) | 1997-08-13 | 1998-08-13 | Lipopolyamines, and the preparation and use thereof |
EP98946333A EP1003711B1 (de) | 1997-08-13 | 1998-08-13 | Neue lipopolyamine, deren darstellung und anwendung |
CA2299429A CA2299429C (en) | 1997-08-13 | 1998-08-13 | New lipopolyamines,their preparation and the use thereof |
DE59802084T DE59802084D1 (de) | 1997-08-13 | 1998-08-13 | Neue lipopolyamine, deren darstellung und anwendung |
AU93421/98A AU745958B2 (en) | 1997-08-13 | 1998-08-13 | Novel lipopolyamines, and the preparation and use thereof |
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DE19735125 | 1997-08-13 | ||
DE19735125.5 | 1997-08-13 | ||
DE19834683A DE19834683A1 (de) | 1997-08-13 | 1998-07-31 | Neue Lipopolyamine, deren Darstellung und Anwendung |
DE19834683.2 | 1998-07-31 |
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EP (1) | EP1003711B1 (de) |
JP (1) | JP4504559B2 (de) |
AT (1) | ATE208369T1 (de) |
AU (1) | AU745958B2 (de) |
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Cited By (5)
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WO1999052858A2 (en) * | 1998-04-08 | 1999-10-21 | Celltech Therapeutics Limited | Lipids |
WO2000064858A1 (en) * | 1999-04-26 | 2000-11-02 | Celltech R&D Limited | Bipolar lipids and their use for the delivery of bioactive substances |
WO2001043778A1 (en) * | 1999-12-17 | 2001-06-21 | Gene Therapy Systems, Inc. | Use of cationic lipids for intracellular protein delivery |
US6852334B1 (en) * | 1999-04-20 | 2005-02-08 | The University Of British Columbia | Cationic peg-lipids and methods of use |
US7189705B2 (en) | 2000-04-20 | 2007-03-13 | The University Of British Columbia | Methods of enhancing SPLP-mediated transfection using endosomal membrane destabilizers |
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US7294511B2 (en) * | 2001-03-22 | 2007-11-13 | Chromos Molecular Systems, Inc. | Methods for delivering nucleic acid molecules into cells and assessment thereof |
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US7906122B2 (en) * | 2003-06-18 | 2011-03-15 | Yissum Research Development Company Of The Hebrew University Of Jersusalem | Sphingoid polyalkylamine conjugates for Hepatitis B virus vaccination |
US20110045001A1 (en) * | 2008-03-28 | 2011-02-24 | Biontex Laboratories Gmbh | Transfection results of non-viral gene delivery systems by influencing of the innate immune system |
WO2013016058A1 (en) * | 2011-07-22 | 2013-01-31 | Merck Sharp & Dohme Corp. | Novel bis-nitrogen containing cationic lipids for oligonucleotide delivery |
CN107405412A (zh) | 2014-11-10 | 2017-11-28 | 埃泽瑞斯公司 | 通过递送bmp编码rna诱导骨生成 |
WO2020165352A1 (en) | 2019-02-14 | 2020-08-20 | Ethris Gmbh | Treatment of ciliopathies |
MX2023005302A (es) | 2020-11-04 | 2023-05-25 | Ethris Gmbh | Uso de arnm de ifn-lambda para el tratamiento de infecciones viricas. |
WO2024121160A1 (en) | 2022-12-05 | 2024-06-13 | Ethris Gmbh | Regulator(s) of energy homeostasis-encoding rna molecule(s) with increased translation efficiency |
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- 1998-08-13 US US09/463,172 patent/US6281371B1/en not_active Expired - Lifetime
- 1998-08-13 AU AU93421/98A patent/AU745958B2/en not_active Ceased
- 1998-08-13 CA CA2299429A patent/CA2299429C/en not_active Expired - Fee Related
- 1998-08-13 WO PCT/EP1998/005156 patent/WO1999008997A1/de active IP Right Grant
- 1998-08-13 EP EP98946333A patent/EP1003711B1/de not_active Expired - Lifetime
- 1998-08-13 ES ES98946333T patent/ES2167939T3/es not_active Expired - Lifetime
- 1998-08-13 JP JP2000509683A patent/JP4504559B2/ja not_active Expired - Fee Related
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Also Published As
Publication number | Publication date |
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JP4504559B2 (ja) | 2010-07-14 |
ATE208369T1 (de) | 2001-11-15 |
ES2167939T3 (es) | 2002-05-16 |
JP2001515060A (ja) | 2001-09-18 |
US6281371B1 (en) | 2001-08-28 |
CA2299429A1 (en) | 1999-02-25 |
AU9342198A (en) | 1999-03-08 |
EP1003711B1 (de) | 2001-11-07 |
CA2299429C (en) | 2010-04-20 |
AU745958B2 (en) | 2002-04-11 |
EP1003711A1 (de) | 2000-05-31 |
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