Classifications

• • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
• • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• • 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
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
• • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
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  • 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
• • 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
  • C07C229/24—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 more than one carboxyl group bound to the carbon skeleton, e.g. aspartic acid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
  • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
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  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
  • C07F9/6503—Five-membered rings
  • C07F9/6506—Five-membered rings having the nitrogen atoms in positions 1 and 3
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/08—Tripeptides
  • C07K5/0827—Tripeptides containing heteroatoms different from O, S, or N
• • 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
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
• • 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
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
  • Y10T428/2991—Coated

Definitions

• the invention relates to amphoteric compounds based on amphiphilic molecules, one or more amphoteric groups having an isoelectric point between 4 and 9 being substituted on their head groups, liposomes containing these compounds and their use.
• lipids summarizes three classes of natural products that can be isolated from biological membranes: phospholipids, sphingolipids and cholesterol with its derivatives.
• Technically produced compounds with similar properties are the diacylglycerols or N, N-dialkylamines.
• liposomes are of technical interest in the production of liposomes.
• These liposomes can be used, among other things, as containers for active ingredients in pharmaceutical preparations. Efficient and stable packaging of the cargo and controllable release of the contents are desirable. It is not easy to combine both claims: the more stable and denser the packaging, the more difficult it is to release the enclosed active ingredient. For this reason, liposomes have been developed that change their properties in response to an external stimulus. Thermosensitive and pH-sensitive liposomes are known. The pH-sensitive liposomes are of particular interest because this parameter can also change under physiological circumstances, for example when a liposome is endocytosed in cells or when the gastrointestinal tract is passed. According to the prior art, pH-sensitive liposomes include in particular cholesterol hemisuccinate (CHEMS).
• CHEMS cholesterol hemisuccinate
• Cholesterol hemisuccinate is used in a mixture with phosphatidylethanolamine to produce pH-sensitive liposomes (Tachibana et al. (1998); BBRC 251: 538-544, US4891208).
• Such liposomes can be endocytosed by cells and in this way are able to transport cargo molecules into the interior of cells without violating the integrity of the cellular membrane.
• a major disadvantage of the CHEMS is its anionic character.
• the liposomes thus produced have a negative total charge and are only taken up by cells with little efficiency. Despite the transfer mechanism described above, they are therefore hardly suitable for the introduction of macromolecules into cells. In addition, it is not possible to package macromolecular agents such as DNA in these liposomes.
• cationic liposomes For the transport of active substances into cells (ransfection), cationic liposomes are used which have a surface charge that is as high and constant as possible. The positive total charge of such particles leads to electrostatic attachment to cells and consequently to efficient transport.
• the use of these compounds and the liposomes produced therewith remains limited to applications in vitro or ex vivo, since such positively charged liposomes form uncontrolled aggregates with serum components.
• WO 00/59474 connections with a membrane anchor and a cationic and anionic head group on the same molecule, the anionic group being connected to the basic structure via a disulfide bridge.
• This disulfide bridge can be reduced under physiological conditions, for example upon contact with the cytosol, the anionic head group is then released and the whole molecule receives a positive charge and enables fusion with the cell membrane.
• the toxicity profile and the storage stability of the compounds disclosed in WO 00/59474 are disadvantageous since free cationic lipids are formed by the elimination of the disulfide bridges. These compounds are known to be disadvantageously cytotoxic.
• the known lipids and the liposomes comprising them can only bind or include below-average amounts of proteins, DNA and / or RNA. If changes are made to the liposomes in such a way that they can bind or include more amounts of carbo, they become cytotoxic or have a low compatibility with serum or blood or are not stable within the blood or serum because they are composed of individual components blood or serum such as complement or perforin.
• the known liposomes and lipids are therefore only suitable to a limited extent for use in the living organism and furthermore have only a low efficiency in the transport of ingredients and / or binding substances. Individual, known compounds which do not have at least some of the disadvantages mentioned can only be produced in a very complicated manner, individual components being so expensive that they cannot be used on a clinical scale or when used in laboratory routines.
• the object was therefore to produce new compounds which do not have the disadvantages mentioned and i) which in particular make it possible to include active substances, in particular plasmids, in liposomes,
• liposomes comprising amphiphilic compounds with an isoelectric point between 4.5 and 8.5, which can reversibly change their charge state from cationic to anionic in a pH range of 1-2 units, according to the general formula (I):
• the amphoter comprises at least one cationic charge part with a pKa value between 4 to 8 and / or at least one anionic charge part with a pKa value between 3 to 7 and optionally further charge carriers, wherein
• the cationic charge part is selected from the group comprising imidazole, morpholine, Piperazine, purine, pyridine and / or pyrimidine or a derivative thereof,
• the anionic charge part is a carboxyl group, the acetic acid, bromoacetic acid, chloroacetic acid bonded in the aliphatic chain,
• Succinic acid maleic acid, fumaric acid, malic acid, tartaric acid, glutaric acid, adipic acid, caprylic acid, pimelic acid, suberic acid, cyclohexanedicarboxylic acid or also cyclopentanedicarboxylic acid; which comprises simply esterified or amidated or in the aliphatic part bound oligocarboxylic acid such as citric acid, isocitric acid or ethylenediaminetetraacetic acid,
• the spacer is a lower alkyl radical with up to 8 C-
• amphiphile is a structure according to the general formula (II) or (III) or (IV):
• Rl and R2 are independently C8 to C30 alkyl or acyl chains with 0, 1 or 2 ethylenically unsaturated bonds and
• Rl and R2 are independently C8 to C30 acyl chains with 0, 1 or 2 ethylenically unsaturated bonds and
• X is -O-.
• Rl and R2 are independently C8 to C30 alkyl chains with 0, 1 or 2 ethylenically unsaturated bonds and
• the amphiphile is a 1,4- or 1,5-dicarboxylic acid esterified with linear C8 to C30 alcohols such as. Aspartic acid, glutamic acid, malic acid, Is tartaric acid, citric acid, aconitic acid, citraconic acid and / or maleic acid and / or
• the amphiphile is a 1,4- or 1,5-diamine of 3-aminoalanine, diaminobutyric acid, ornithine or amidated with linear C8 to C30 fatty acids
• the invention thus relates to the teaching that by conjugating amphoteric groups via a spacer to an amphiphile that can be incorporated into liposomal membranes, structures can be provided that can be used in particular for the production of liposomes that are suitable for use in the living organism to become and can bind and / or transport high amounts of proteins, peptides, carbohydrates, DNA and / or RNA.
• the new compounds are suitable for producing vesicles or liposomes which do not aggregate in the blood or serum, which are not attacked by complement components, which are not cytotoxic and which are stable in the blood or serum for several hours, the permeability of the liposomes from the pH Value and thus depends on the charge status of the connections. That The active ingredient releases of these non-aggregating, stable, non-cytotoxic liposomes are dependent on the pH of the medium.
• the one or two long-chain alkyls or acyls present in this molecular building block comprise between 8 and 30 carbon atoms. They are preferably straight-chain or little branched and can have 0.1 or 2 ethylenically unsaturated bonds. Those substituents which are found in natural lipids, ie straight-chain fatty acids or alcohols having 12 to 20 carbon atoms and none, one or two unsaturated bonds, are particularly preferred. Lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl and linoyl residues, or their corresponding fatty alcohols, are very particularly preferred.
• Preferred amphiphiles are diacylglycerols, dialkylglycerols, phosphoglycerols, acylated or alkylated 3-amino-1,2-propanediols or else the N, N-dialkylamines, since these compounds are particularly cheaply available, have simple chemistry and are present in a high proportion Membranes can be installed without increasing their permeability or even completely destroying the membrane character.
• dicarboxylic acids are used as the polar head group of the amphiphilic, which expediently allow coupling of the ultimately charged substituents via further functional groups.
• amphiphiles derived therefrom are: long-chain esters of 1,4- or 1,5-dicarboxylic acids, such as aspartic acid, Glutamic acid, malic acid, tartaric acid, citric acid, aconitic acid, citraconic acid, maleic acid or similar such compounds with fatty alcohols.
• the lauryl, myristyl, palmityl, stearyl, oleyl and linole esters of the dicarboxylic acids mentioned are particularly preferred.
• Other molecular building blocks spacer, amphoter) are coupled via the remaining amino group, hydroxyl group, carboxyl group or via the double bond.
• amphiphiles are obtained from diamines with a further functional group, for example as the diamide of 3-aminoalanine, diaminobutyric acid, ornithine or lysine with long-chain fatty acids.
• diamines with a further functional group for example as the diamide of 3-aminoalanine, diaminobutyric acid, ornithine or lysine with long-chain fatty acids.
• the ' lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl and linoyl radicals are particularly preferred.
• the compounds according to the invention can also be prepared as derivatives of sphingosine or ceramides. They can also be represented as derivatives of long-chain vinyl ethers or plasmalogens.
• amphiphiles which are advantageously used as starting material, can be functionalized differently on their hydrophilic head group in order expediently to permit a permanent, but also biodegradable coupling, or optionally to perform the function of a spatial spacer.
• the hydroxyl group or an amino group present are particularly suitable for direct coupling.
• Carboxyl groups are also suitable.
• Amphoter The whole molecule receives its pH-dependent charge characteristic through the simultaneous presence of cationic and anionic groups in the molecular part "Amphoter".
• An amphoter is particularly characterized in that at a certain pH the sum of its charge components is just zero. This point is called the isoelectric point (iP). Above the iP the connection is negatively charged, below the iP it is to be regarded as a positive cation; wherein the iP of the compounds according to the invention is between 4.5 and 8.5.
• the total charge of the molecule at a certain pH of the medium can be calculated as follows:
• a compound according to the invention is formed, for example, by coupling the amino group of histidine to dipalmitoylglycerol hemisuccinate.
• the product is negatively charged at a neutral pH of 7 because the carboxyl function is essentially completely dissociated and the imidazole function is only slightly charged.
• an acidic pH of about 4 the situation is reversed: the carboxyl function is now largely discharged, but the imidazole group is essentially completely protonated, and the overall charge of the molecule is therefore positive.
• a compound that is intended to clarify the inventive teaching is created by coupling histidine to phosphatidylserine. It is irrelevant whether the coupling takes place between the carboxy group of the histidine and the amino group of the PS or whether the amino group of the histidine is coupled to the carboxyl function of the PS. In both cases, molecules are formed in which a free amino group neutralizes the constant negative charge of the phosphate group. The remaining carboxyl group and the imidazole function react as above, so that the intended characteristic occurs before the structures according to the invention.
• the molecule has an isoelectric point between 4 and 8, preferably between 5 and 7.
• the amphoter comprises one or more cations with a pKa value between 4 and 8 and at the same time one or more anions with a pKa value between 3 and 7.
• Amphoters can in particular be composed of two charge carriers, both of which change their charge in the specified pH range between 'and 9. The simultaneous loss of anionic charge and gain of cationic charge leads to a charge change of the whole molecule.
• a particularly preferred embodiment comprises amphoterics in which the pKa values of cation and anion are at most 2 pH units apart. This has a particularly advantageous effect on the sharpness of the iP. The closer the two pKa values are to each other, the narrower the pH range in which the molecular charge is from cationic to changes anionically.
• the person skilled in the art can make an advantageous selection of the type and number of cations and anions using the above-mentioned formula.
• these ( fixed charges of the amphoter must be overcompensated for by the variable charges described. This is only possible if the variable charge carriers are used in excess. If, for example, a tertiary amine is used as the cation, at least 2 carboxy groups are required to form an amphoter to obtain the purposes of the invention. in only one of the carboxyl group can 'amine only be compensated, the positive charge, the molecule never completely reload. advantage of using fully dissociated groups is their strong polarity.
• Amphoters can particularly preferably be present as complete structural units. This is preferably the case with o-, m- or p-aminobenzoic acids, imidazole carboxylic acid, imidazole diacetic acid or also nicotinic acid or picolinic acid. Further advantageous compounds are, for example, w- (1-piperazino) alkylcarboxylic acids, urocanoic acid, 4- (2-aminoethyl) imidazole-maleic acid monoamide, 4- (2-hydroxyethyl) imidazole-maleic acid monoester, (2-aminoethyl) morpholine-maleic acid monoamide or analog connections.
• pH-sensitive cations with a pKa between 4 and 8 pH-sensitive cations with a pKa between 4 and 8:
• the cation is preferably an imidazole, piperazine, morpholine, purine or pyrimidine.
• Other advantageous cations with this property are essentially nitrogen bases.
• the nitrogen bases are present as ring systems, positional isomers advantageously exist in which the connecting spacer is substituted at different positions of the organic cation.
• the pKa values of the organic cations can expediently be influenced by the position isomerism.
• the underlying rules are known to the person skilled in the art. Alternatively, these influences can be estimated from tables (Handbook of Chemistry and Physics, 73rd volume, pp. 8 - 37 ff.).
• the piperazines, imidazoles and morpholines, purines or pyrimidines mentioned are particularly preferred.
• Molecular fragments such as those found in biological systems are very particularly preferred, for example 4- imidazoles (histamines, histidine themselves), 2-, 6- or 9-purines (adenines, guanines, adenosines or guanosines), 1-, 2- or 4-pyrimidines (uraciles, thymines, cytosines, uridines, thymidines, cytidines) or pyridine-3-carboxylic acids.
• the structural fragments mentioned here can have further substituents.
• These can be, for example, methyl, ethyl, propyl or isopropyl radicals, particularly preferably in hydroxylated form with one or two hydroxyl groups. However, this can also be hydroxyl or keto functions of the ring system.
• Nitrogen bases with preferred pKa values are also formed, for example, by single or multiple substitution of an amine with lower alkane hydroxyls, for example hydroxymethyl or hydroxyethyl groups.
• Suitable organic bases from this group are, for example, aminopropanediols, triethanolamines, tris (hydroxymethyl) methylamines, bis (hydroxymethyl) methylamines, tris (hydroxyethyl) methylamines, bis (hydroxyethyl) methylamines or the correspondingly substituted ethylamines.
• Nitrogen bases with preferred pKa values can also be found among the amino sugars or amino sugar alcohols.
• the anionic charge carriers are preferably carboxyl groups.
• carboxylic acids can be used as charge carriers. These include in particular aliphatic, straight-chain or branched carboxylic acids with up to 8 carbon atoms and 0, 1 or 2 ethylenically unsaturated bonds.
• Exemplary connecting parts are the carboxyl group itself, the acetic acid, bromoacetic acid, chloroacetic acid, acetoacetic acid, propionic acid, acrylic acid, butyric acid, crotonic acid or higher carboxylic acid bound in the aliphatic chain, the simply esterified or amidated or bonded in the aliphatic chain, such as oxalic acid, malonic acid, succinic acid , Maleic acid, fumaric acid, malic acid, tartaric acid, glutaric acid, adipic acid, caprylic acid, pimelic acid, suberic acid, cyclohexanedicarboxylic acid or also cyclopentanedicarboxylic acid; the simply esterified or amidated or in the aliphatic part bound oligocarboxylic acid such as citric acid, isocitric acid or ethylenediaminetetraacetic acid.
• connecting parts are the glycolic acid, lactic acid, hydroxybutyric acid, malic acid, tartaric acid, aspartic acid or glutamic acid, alanine, glycine, serine, threonine, asparagine, glutamine, proline, tyrosine or cysteine or other amino acids or hydroxy acids bound in the side chain via a hetero atom.
• Carboxylic acids with a suitable behavior can also be found as substituents in aromatic systems, for example as benzoic acid, anisic acid, o-, m- or p-hydroxybenzoic acid, as dihydroxybenzoic acid, gallic acid, cinnamic acid,
• Other anionic groups are dissociable hydroxyls or thiols, as they occur in ascorbic acid, the N-substituted alloxan, the N-substituted barbituric acid, in the veronal, the phenol or as a thiol group.
• Peptides as amphoteric In a preferred embodiment of the invention, the amphoteric are peptides and comprise 2 to 6 amino acids.
• amino acids histidine, arginine, lysine, glutamic acid or aspartic acid are particularly preferred for the formation of the amphoteric and for determining its charge characteristic.
• Other preferred amino acids are glycine, serine, threonine, glutamine, asparagine, but also cysteine, which contribute to increasing the polarity and thus improving the solubility of the amphoteric.
• Table 1 shows particularly preferred compositions of the peptides in percentage of total amino acid:
• the spacer is a lower alkyl radical with a linear, branched or ring-shaped structure, which has 0 to 8 C atoms and contains 0, 1 or 2 ethylenically unsaturated bonds ,
• the spacer can have hydroxyl groups to increase the polarity of the molecule.
• the spacer can in particular be a sugar.
• Spacer may advantageously also be a polyethylene glycol ', this may comprise up to 20 monomer units.
• X and / or Y can also be deletions, i.e. their presence is not mandatory.
• Group Y can be omitted, for example, if the amphoter can be coupled directly to the amphiphile, for example in the esterification of imidazole-4,5-dicarboxylic acid with dipalmitoylglycerol.
• Synthesis methods Methods for carrying out the chemical coupling of the individual molecular building blocks are known to the person skilled in the art and can vary depending on the starting material and coupling component used. Typical reactions are esterification, amidation, the addition of amines to double bonds, etherification or reductive amination.
• Particularly preferred molecules can be produced by i) esterification of diacylglycerols,
• the particularly preferred compounds include the following embodiments (the long-chain hydrocarbon chains of the amphiphiles are only given in abbreviated form and correspond to lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl and linoyl residues):
• long-chain amphiphiles are only given in abbreviated form and correspond to lauryl, myristyl, palmityl, stearyl, oleyl and linyl residues.
• the particularly preferred amphoteric components include, for example, the following compounds, where R1 or R2 are the amphiphile and () n are further parts of the molecule in the sense of the spacer defined above.
• amphiphile can be coupled via one of the ring atoms.
• the preferred derivative shown here shows the coupling of piperazine to the N of the phosphatidylserine.
• amphiphile can be coupled via one of the ring atoms.
• the side chains as hydroxycarboxylic acids or amino acids carried out, the coupling can advantageously take place via these heteroatoms.
• the amphiphile is preferably bound as an ester of one of the two acetic acid functions.
• the amphiphile can also be linked to the 3-amino function. Derivatives of piperidine.
• amphiphile can be coupled via one of the ring atoms. If the side chains are designed as hydroxy or amino acids, then the Coupling takes place extensively via their heteroatoms.
• amphiphiles are preferably coupled via one of the two amino groups.
• the second amino group can, for example, be alkylated in order to obtain a higher pKa.
• Coupling as an amide of phosphatidylserine is another preferred embodiment of the invention.
• Amphoteric groups also result from the esterification of trinitriloacetic acid.
• the charge behavior of these compounds can also be changed by complexing metal ions.
• Amphoteric compounds are also formed here by coupling sterols to the terminal groups of N-acylamino acids.
• the structure can be advantageous from the serine Aspartic acid or glutamic acid or derived from lysine or ornithine.
• the aminodicarboxylic acids can not only end, but also on be coupled to the other acid groups.
• the charge behavior of these compounds can also be changed by complexing metal ions.
• Amphoteric groups also result from the esterification of trinitriloacetic acid.
• the charge behavior of these compounds can also be changed by complexing metal ions.
• the invention also relates to liposomes comprising the compounds according to the invention.
• a large proportion of the compounds according to the invention can be incorporated into liposomal membranes and form amphoteric liposomes which are characterized in that their charge state changes reversibly due to a change in the pH of the surrounding medium.
• the liposomes are cationic below their isoelectric point and anionic above.
• Liposomes comprising the compounds according to the invention can be coated with polymers under suitable conditions. Single or multiple deposition of such substances can take place on the surface. A multiple deposition, which may be carried out in the presence of a crosslinking agent, results in liposomal nanocapsules.
• the liposomes are only sterically enclosed in the nanocapsules, and the membrane no longer interacts with the polyelectrolytes. Clustering of the lipids and associated permeabilization of the membrane can thus advantageously be avoided.
• liposomes the membranes of which contain the substances according to the invention, easily fuse with other membranes, in particular cell membranes, below the isoelectric point of the substance.
• this step requires the presence of a greater proportion of PE in the membrane. Due to its tendency to form hexagonal phases, this has the function of a helper lipid.
• the disadvantage is the lower stability such membranes, a gradual release of trapped active substances is often observed here.
• Liposomes that are produced using the substances according to the invention advantageously fuse effectively even in the absence of helper lipids. Liposomes can thus be produced using the substances according to the invention which can stably encapsulate an active ingredient, but fuse under the conditions of a low pH with cell membranes and release the active ingredient there.
• the proportion of the amphoteric lipids comprises a maximum of 30 mol%, 40 mol% or 50 mol% of the total lipid.
• Compositions which comprise at least 2 mol% but at most 50 mol% of the amphoteric lipids are particularly advantageous.
• Compositions which comprise at least 5 mol%, preferably 10 mol% and at most 50 mol%, preferably 40 mol% of the amphoteric lipid are particularly preferred.
• Liposomes comprising the substances according to the invention are prepared by the techniques known to the person skilled in the art.
• the liposomes comprise phosphatidylcholine, phosphatidylethanolamine, diacylglycerols, ceramides, sphingolipids, tetraether lipids and / or PEG lipids. Since the compounds according to the invention do not always form liposomes themselves, it may be advantageous to add the lipids mentioned.
• the liposomes have an average size between 50 and 1000 nm, preferably between 50 and 500 nm, particularly preferably between 50 and 300 nm and very particularly preferably between 60 and 130 nm.
• Liposome dispersions can be injected, infused or implanted. They then distribute themselves in the blood or in the lymph or give off their active ingredient in a controlled manner as a depot. The latter can be achieved by highly concentrated dispersions, which are available as gels.
• the liposomes can also be used for topical application on the skin. In particular, they can contribute to the fact that various active ingredients can penetrate the skin better or even get into the body through the skin. It is also possible to use the liposomes for gene transfer. Because of its size and charge, genetic material cannot usually get into cells without aids.
• the active substance is a protein, a peptide, a DNA, an RNA, an antisense nucleotide and / or a decoy nucleotide.
• the basic structure of liposomes is very similar to that of cell membranes. They can therefore be used as membrane models to quantify the rate of permeation of active substances through membranes or the membrane binding of active substances.
• At least 50 ⁇ g, advantageously 100 / xg, preferably 150 / xg of the active ingredient per mg lipid are included in the liposomes.
• Cargomolecules that are not installed and adhere to the outside can, however be removed by simply increasing the pH. This step is always necessary if non-built-in cargo molecules lead to an aggregation of the liposomes.
• An advantage of using the components according to the invention is the fact that the enclosed active substances only have to be brought under conditions that permit interaction with the lipid layer for the period of the actual inclusion. As soon as the lipid layer remains closed, you can switch to other conditions. A possible inactivation of active ingredients, especially proteins, can be minimized.
• the invention also relates to methods for loading liposomes with active substances, wherein a binding pH value is used for encapsulation and a second pH value is used for separating the unbound active substances.
• the invention further relates to a method for loading active ingredients into liposomes, the liposomes being permeabilized at a specific pH and subsequently sealed.
• changes in permeability can be used specifically for loading liposomes.
• An active substance to be enclosed can be added to the medium under conditions of high permeability. Conditions of low permeability are then set. The active ingredient thus remains inside the liposomes. Active substance not included can then optionally be separated off.
• Such a change in permeability can be brought about on liposomes or on liposomal nanocapsules.
• the invention also relates to the use of the liposomes in diagnostics and in release systems.
• the liposomes can of course also be used in a detection system.
• the liposomes can Metal ions are loaded, the fluorescence of which is enhanced by the chelation, for example terbium or europium ions.
• Liposomes for this application can of course contain specificity-determining components, for example antibodies, lectins, selectins, receptors or hormones or RNA aptamers.
• the presence of these metal ions is restricted to the lumen of the liposomes in order to avoid unspecific signals from metal ions which are adhering and slowly released. It is also expedient to use the liposomes for the production of nanocapsules.
• the liposomes can advantageously be used for the production of release systems in diagnostics.
• the liposomes can advantageously be used as a depot formulation and / or as a circulable depot. It is also advantageous to use the liposomes as vectors for transfecting cells in vivo, in vitro and / or ex vivo.
• the liposomes can be used, for example, with intravenous and / or peritoneal application.
• the compounds and liposomes according to the invention have several advantages. Surprisingly, it was found that the permeability of the liposomes according to the invention depends on the pH and thus on the charge state of the compounds.
• Liposomes which are produced using the structures according to the invention are therefore particularly suitable for the construction of release systems in which active substances should be released depending on the pH of the medium.
• above-average amounts at least 50 ⁇ g, preferably 10 ⁇ g, are particularly preferred in liposomes, the membrane of which comprises the compounds according to the invention.
• 150 ⁇ g of proteins or DNA per mg of lipid can be included.
• the efficiency of this installation depends on the pH of the solution used.
• a process for the efficient encapsulation of proteins or DNA into the liposomes can therefore be carried out by first setting a pH value which leads to a good binding of the cargo molecules to the lipid layer.
• a pH value which leads to a good binding of the cargo molecules to the lipid layer.
• the usable pH value depends on the isoelectric point of the protein. This should be below the isoelectric point of the substance according to the invention.
• Encapsulation is particularly effective if the pH of the medium is chosen so that it lies between the isoelectric point of the protein and the isoelectric point of the compound according to the invention. The protein is then negative and the lipid layer is positively charged.
• liposomes the membrane of which, for example, comprises histidinyl PS or histidinyl diacylglycerol hemisuccinate
• This property leads to an increase in the positive charge of the liposome. This effect can be observed particularly strongly at neutral pH values, since the self-loading of the compound is then low. Due to their chelating properties, such liposomes can be used in biochemical diagnostics and pharmaceutical therapy.
• An essential prerequisite for the use of liposomes for experimental or therapeutic purposes is their compatibility with cells and tissues.
• a number of known compounds which are used for the introduction of DNA or proteins into cells for example the cationic lipid DOTAP
• cytotoxic A number of known compounds which are used for the introduction of DNA or proteins into cells (for example the cationic lipid DOTAP) are cytotoxic. It has surprisingly been found that some of the compounds according to the invention show reduced cytotoxicity. This applies in particular to the group of compounds in which the amphoteric is an amino acid or a peptide. These therefore
• liposomes are attacked by the components of the complement system and quickly lysed. This reaction takes place within minutes. Pore formation occurs in the membrane, through which even large molecules such as proteins can diffuse out. Stabilization of liposomes against these mechanisms has so far only been possible by incorporating cholesterol into the lipid layer. Such liposomes are then very stable, but can no longer interact with cells or release their active ingredient easily. It has surprisingly been found that liposomes which are built up using the components according to the invention can be stable in serum or blood for several hours. The drug release is low even under these conditions.
• a liposomal vector for the transport of active substances must meet at least three requirements: it must have low toxicity, contain the active substance safely and stably and be compatible with serum or blood.
• liposomes are produced using selected substances according to the invention, fulfilled with advantage.
• the liposomes are therefore well suited for use for therapeutic purposes. Further properties that support this use are the good loading capacity with active substances and the targeted detachment of these substances by changes in pH -value or by permeabilization of the membrane.
• liposomes prepared using the substances of the invention show low non-specific binding to cell surfaces. This low nonspecific binding is an essential condition for the realization of 'a specific binding to target cells. If the liposomes described The vehicle can then be specifically enriched in those cells or tissues that have pathological conditions.
• An essential use of the substances according to the invention therefore lies in the construction of vectors for the transfer of active substances in living organisms.
• the vectors are Particularly suitable for the transport of therapeutic macromolecules such as proteins or DNA, which cannot inherently cross the cell membrane or which are rapidly broken down in the bloodstream.
• amphoteric amphiphiles with only one hydrocarbon chain are also suitable for the production of amphoteric liposomes according to the invention if the hydrocarbon chain comprises more than 12 CH2 groups.
• These liposomes can advantageously be loaded with active substance, in particular DNA or oligonucleotides, by the above-mentioned method, and their use for transfection of cells and for gene therapy is included in the disclosure of the present invention.
• two-chain amphiphiles which are formed by amidation of long-chain fatty acids with long-chain ⁇ -amino-carboxylic acids and are provided with an amphoteric group, such as histidine, are also suitable for the production of amphoteric liposomes.
• These liposomes can advantageously be loaded with active substance, in particular DNA or oligonucleotides, by the above-mentioned method, and their use for transfection of cells and for gene therapy is included in the disclosure of the present invention.
• the DG-Hist-Succ is synthesized in three stages. First, 3.7 mmol (2 g) of dipalmitoylglycerol are esterified with 4.1 mmol of amino-protected CBZ histidine. The ester formation takes place with the addition of 4.3 mmol (0.67 g) EDC and 4.3 mmol (0.53 g) DMAP in 60 ml dichloromethane. The mixture is stirred for 4 h. Deprotection of the amino function of histidine produces the intermediate product DG-hist. The amino group is deprotected by catalytic hydrogenolysis on 10% palladium / carbon by stirring overnight under a hydrogen atmosphere.
• succinic anhydride is opened to the benzyl succinate with benzyl alcohol.
• 10 mmol (1 g) of succinic anhydride are dissolved in 9.5 ml (1 g) of benzyl alcohol in 50 ml of toluene.
• 1 mmol (190 mg) of p-toluenesulfonic acid monohydrate the mixture is heated under reflux for two hours.
• 2 mmol (0.42 g) benzyl-protected succinate is coupled to 1.6 mmol (1.13 g) DG-Hist via an amide bond.
• the desired compound is synthesized in three stages.
• Unilamellar liposomes (DPPC / DPPG / cholesterol 40:20:40 mol%) are suspended in a concentration of 20 mM lipid in a borate buffer (20 mM sodium borate, 120 mM sodium chloride pH 8.4). 400 ⁇ l of a 0.6M sodium periodate solution are added to 2ml of this solution, the mixture is incubated for 30min in the dark. 1ml of such a suspension is chromatographed on Sephadex G25 in the borate buffer used above. The eluate of the liposome suspension is made up to 4 ml. Carnosine is added to the liposomes thus oxidized in a final concentration of 20 mM and incubated for 2 hours. Finally, it was reduced with 20 mM sodium borohydride overnight at 4 ° C. Excess carnosine can be separated by chromatography on Sephadex G25 as above. Example 6
• the lipid film is dried in vacuo overnight.
• the lipid film is hydrated directly with 1 ml of DNA-containing (100 ⁇ g DNA / ml) NaAc buffer (10 mM NaAc, 150 mM NaCl, pH 4) (light ultrasound, then for 30 min above the
• Phase transition temperature rotates). Then there is a freeze / thaw step.
• the mixture is extruded 15 times through 400 nm membranes 10 ° C above the phase transition temperature.
• DNA that is not included can be flotated in the sucrose
• the DNA content is determined using the intercalation dye
• Propidium iodide is determined by the increase in fluorescence intensity when intercalated into the DNA. For this, 20 ⁇ l
• Triton X-100 10% in water made up to 300 ⁇ l with sample and measured with a fluorescence plate reader.

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