EP1347964A2 - Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats lipidiques ainsi que leur utilisation - Google Patents

Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats lipidiques ainsi que leur utilisation

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Publication number
EP1347964A2
EP1347964A2 EP01985932A EP01985932A EP1347964A2 EP 1347964 A2 EP1347964 A2 EP 1347964A2 EP 01985932 A EP01985932 A EP 01985932A EP 01985932 A EP01985932 A EP 01985932A EP 1347964 A2 EP1347964 A2 EP 1347964A2
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EP
European Patent Office
Prior art keywords
tetraether
nhco
lipid
lipid derivative
derivative according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP01985932A
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German (de)
English (en)
Inventor
Christine KÜHL
Bernhard Tewes
Martin Hagen
Felix Gropp
Ralf Littger
Uwe Marx
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Bernina Biosystems GmbH
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Bernina Biosystems GmbH
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Application filed by Bernina Biosystems GmbH filed Critical Bernina Biosystems GmbH
Publication of EP1347964A2 publication Critical patent/EP1347964A2/fr
Withdrawn legal-status Critical Current

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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—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D323/00—Heterocyclic compounds containing more than two oxygen atoms as the only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/10—Phosphatides, e.g. lecithin

Definitions

  • the present invention relates to tetraether lipid derivatives, the liposomes and lipid agglomerates containing the tetraether lipid derivatives according to the invention and their use.
  • Liposomes are artificially produced uni- or multilamellar lipid vesicles that enclose an aqueous interior. Compounds possibly contained in the aqueous interior of the liposome are largely protected against proteolytic or nucleolytic attacks.
  • the lipid vesicles are generally similar to biological membranes and are therefore often easily integrated into the membrane structure after attachment to them. With this membrane fusion, the contents of the liposome interior are discharged into the lumen enclosed by the biological membrane. Alternatively, the liposomes are broken down in the cell lysosomes after endocytotic uptake. The content of the liposome interior released there can then pass from there into the cytosol of the cell.
  • Liposomes can therefore be used as transport vehicles.
  • liposomes are widely used as transport vehicles for therapeutic agents.
  • hydrophilic therapeutic agents for example “small molecules”, peptides or proteins are packaged in the aqueous interior of the liposome and / or hydrophobic compounds are built into the hydrophobic matrix, ie the lipid layer of the liposomes .
  • the cosmetics industry produces liposome-containing skin creams that transport active ingredients into the epidermis and lower cell layers.
  • targeted delivery of the active substances transported by the liposomes to specific cells or their accumulation in the vicinity of such target cells can be achieved, for example, by antibodies coupled to the outer membrane of the liposomes for certain cell surface structures or other “targeting” units.
  • Mainly natural lecithins from soybeans or egg yolk or defined natural or artificial phospholipids such as cardiolipin, sphingomyelin, lysolecithin and others are used to produce liposomes.
  • polar head groups choline, ethanolamine, serine, glycerol, inositol
  • the length and the degree of saturation of the hydrocarbon chains, size, stability and ability to take up and release the associated molecules are influenced.
  • Liposomes formed from normal double-layer-forming phospholipids can only be kept for a short time even when cooled.
  • Their storage stability can be e.g. by including phosphatidic acid or ⁇ -tocophero, but the stability thus improved is still insufficient for many purposes.
  • conventional liposomes are not acid-stable and are therefore unsuitable for the transport of active pharmaceutical ingredients that pass through the stomach after oral administration, nor for liposome-assisted DNA transfection under slightly acidic pH conditions.
  • Liposome-forming lipid mixtures for example Lipofectamin®, Lipofectin® or DOTAP®, are frequently used for scientific and medical lipofections in mammalian cells.
  • their use also means that a large number of parameters (eg cell density, amount of nucleic acid, Proportion of lipids added, volume of liposome preparation, etc.) to be determined exactly. because there is only a very narrow parameter optimum at which sufficient transfection efficiencies can be achieved. This makes transfections using commercial lipofection reagents very complex and cost-intensive.
  • large variations between the individual batches can be observed in the products mentioned above, which makes them less reliable in practice.
  • WO-A-97/31927 (DE-A-19607722) describes a tetraether lipid derivative which comprises a side chain with a modified or unmodified gulose residue or an oxidation product of such a gulose, and liposomes containing this tetraether lipid derivative, which are characterized by improved resistance distinguished from acids and by an improved storage stability.
  • US Patent 5098588 describes the preparation of tetraether lipid derivatives with polar side chains and their use as an interface lubricant.
  • WO-A-99/10337 (DE-A-19736592) describes tetraether lipid derivatives with side chains which are positively charged either per se through the formation of quaternary ammonium salts or under physiological conditions.
  • Such derivatized lipids are suitable for contacting negatively charged molecules, e.g. nucleic acid molecules, and e.g. to be enclosed in liposomes.
  • lipids which enable the production of liposomes which are improved with regard to their in vivo stability or storage stability.
  • lipids which, in addition to increased stability, offer an improved possibility of optionally delivering active substance from the liposomes integrating them.
  • the object of the invention is therefore to provide lipids which are suitable for the formation of liposomes and which have improved stability and / or allow a targeted release of active ingredient.
  • TEL derivatives tetraether lipid derivatives
  • X 1 and X 2 independently of one another are a branched or unbranched alkylene or alkenylene group having 1 to 20 carbon atoms, -COCR 1 Y (CH 2 ) m -, - (CH 2 ) m NHCO-, - (CH 2 ) m NHCOCHY-, - (CH 2 ) m O (CH 2 ) m NHCOCHY-, - [(CH 2 ) m O] n -, - [(CH 2 ) m O] n CO-, - (CH 2 ) m NHCO (CH 2 ) m CO-, - (CH 2 ) m -, - (CH 2 ) m CO-, - (CH 2 ) m NH- or - (CH 2 ) m NHCO (CH 2 ) m -,
  • R 1 and R 6 -H a straight-chain, branched or cyclic alkyl, alkenyl, aralkyl or aryl group with 1 to 12 carbon atoms, where these groups can be substituted with at least one group Y, or- (CH 2 ) m NHR 2 ,
  • n 0 to 150 o 0 to 10
  • p 0 or 1 q 0 to 5 and r 0 or 1
  • radicals R 2 to R 5 , R 7 and R 8 can further comprise a ligand which can be bound to the lipid via a spacer, and modifications thereof formed by the formation of pentacycles in the basic tetraether structure,
  • the present invention also provides liposomes and lipid agglomerates containing at least one of the aforementioned tetraether lipid derivatives.
  • the lipid structure of the cyclic tetraether lipid derivatives according to the invention consists of a 72-membered macrotetraether cycle. This consists of two biphytanyl chains, the terminal carbon atoms of which are linked to one another via two ethylenedioxy units (-O-CH 2 -CH 2 -0-). One carbon atom of each ethylene unit is substituted with a group S. Tetraether lipids are already known and have so far only been found in archaebacteria. Depending on the cultivation temperature, for example, pentacycles can form within the biphytanyl chains, giving the lipid a specific physico-chemical character. With every pentacyclization, the backbone loses two hydrogen atoms.
  • lipid scaffold has now been derivatized according to the invention in order to be suitable for incorporation into liposomes or lipid agglomerates intended for transfection or for the delivery of active substance with improved properties with regard to transfection and stability.
  • special side chains are introduced.
  • Some of the lipids derivatized in this way are particularly well suited to come into contact with negatively charged molecules, for example nucleic acid molecules, and to enclose them, for example, in liposomes.
  • the lipids according to the invention can additionally be coupled with molecules which enable the lipids to be specifically docked onto special cells. Examples of these are antibodies against cell surface antigens, in particular those which are selectively expressed on the target cells. Also possible are ligands for receptors that can be found selectively on the surface of certain cells, as well as biologically active peptides that enable organ or cell-specific targeting in vivo (Ruoslati, Science, 276, 1345-46, May 30, 1997) ,
  • the particular advantage of the tetraether lipid derivatives according to the invention is their improved stability. Since the lipid structure of the tetraether lipid derivatives according to the invention contains no double bonds, they are insensitive to oxidation. Furthermore, instead of the lipid ester bonds contained in lipids from eubacteria and eukaryotes, the tetraether lipid derivatives according to the invention only contain lipid ether bonds which are also present at high proton concentrations, such as those e.g. occur in the stomach, not be attacked. Another advantage of the tetraether lipids is the membrane-spanning macrocycles, which lead to better anchoring of the lipid in the membrane and thus increase the stability of the liposomes.
  • the tetraether lipid derivatives according to the invention preferably contain 0 to 8 pentacycles in the tetraether backbone.
  • Examples of such tetraether backbones include the following macrocycles:
  • the stereocenters of the biphytanyl chains of the tetraether macrocycles are preferably in the configuration shown above.
  • the glycerol units preferably have an sn configuration.
  • X 1 and X 2 independently of one another are a branched or unbranched alkylene or alkenylene group having 1 to 20 carbon atoms, -COCR 1 Y (CH 2 ) m -, - (CH 2 ) m NHCO-, - (CH 2 ) m NHCOCHY-, - (CH 2 ) m O (CH 2 ) m NHCOCHY-, - [(CH 2 ) m O] n -, - [(CH 2 ) m O] n CO-, - (CH 2 ) m NHCO (CH 2 ) m CO-, - (CH 2 ) m -, - (CH 2 ) m CO-, - (CH 2 ) m NH- or - (CH 2 ) m NHCO (CH 2 ) m -,
  • R 1 and R 6 -H a straight-chain, branched or cyclic alkyl, alkenyl, aralkyl or aryl group with 1 to 12 carbon atoms, where these groups can be substituted with at least one group Y, or- (CH 2 ) m NHR 2 ,
  • radicals R 2 to R 5 , R 7 and R 8 can further comprise a ligand which can be bound to the lipid via a spacer.
  • S 1 and S 2 independently of one another represent one of the following groups:
  • R 1 to R 6 independently of one another -H, -CH 3 , -C 2 H 5 ;
  • n is an integer from 1 to 4 and s is an integer from 1 to 4.
  • Preferred combinations of S 1 and S 2 include:
  • Preferred groups represented by D include - (CH 2 CH 2 0) n -E, - (CH 2 ) 3 -Y, - (CH 2 ) 3 -NHCO-CHY- (CH 2 ) 3 -Y, - (CH 2 ) 2 -N [(CH 2 ) 2 -Y] 2 and - (CH 2 ) -NR 2 - (CH 2 ) 3 -Y.
  • Particularly preferred groups represented by D include - (CH 2 ) 3 -N (R 5 ) 2 , - (CH 2 ) 3 -N + (R 5 ) 3 , - (CH 2 CH 2 0) n - CH 3 , - (CH 2 ) 3-NHCO-CH [N (R 5 ) 2] - (CH2) 3-N (R 5 ) 2 , - (CH2) 2-N [(CH 2 ) 2-N ( R 5 ) 2 ] 2 , - (CH 2 ) 2 -N [(CH 2 ) 2 -N + (R 5 ) 3 ] 2 , - (CH 2 ) 4 -NH- (CH 2 ) 3 -N (R 5 ) 2 and - (CH 2 ) 4 -NH- (CH 2 ) 3 -N + (R 5 ) 3 .
  • Preferred groups represented by E include - (CH 2 ) 2 -Y, -CH 3 , - (CH 2 ) 2 -NHC0-Z, - (CH 2 ) 2 -NR 4 ligand and - (CH 2 ) m -CO ligand.
  • Preferred groups represented by Y include -NHR 5 , -N (R 5 ) 2 , -N + (R 5 ) 3 and -NH-C (NH) -NH 2 , the groups -NH 2 , - N (CH 3 ) 2 and -N + (CH 3 ) 3 are particularly preferred.
  • Preferred groups represented by Z include: - (CH 2 ) 2 - CO ligand,
  • the groups X 1 and X 2 are preferably straight-chain alkylene groups with 1 to 12, preferably with 1 to 6 and particularly preferably with 1 to 4 carbon atoms. Examples of preferred groups include - (CH 2 ) -, - (CH 2 ) 2 -, - (CH 2 ) 3 - and - (CH 2 ) 4 -.
  • Preferred groups represented by R 1 and R 6 include hydrogen and alkyl groups having 1 to 6, particularly preferably 1 to 3 carbon atoms, such as a methyl group, an ethyl group or an n-propyl group, which is substituted with at least one group Y. could be.
  • Particularly preferred examples of the substituted and unsubstituted alkyl groups represented by R 1 and R 6 include -CH 3 , -C 2 H 5 , - (CH 2 ) 3-N (CH 3 ) 2 and - (CH 2 ) 3 -NH 2 .
  • m is preferably a number in the range from 1 to 3, particularly preferably 2 or 3.
  • n is preferably a number in the range from 3 to 130 and very particularly preferably a number in the range from 40 to 85.
  • o is preferably a number in the range from 0 to 3, particularly preferably 0 or 1.
  • q is preferably 0 or 1.
  • S 1 and S 2 are independently selected from the following groups:
  • S 1 and S 2 are very particularly preferred combinations of S 1 and S 2.
  • one of the radicals R 2 to R 5 , R 7 and R 8 can further comprise a ligand which can be bound to the lipid via a spacer.
  • the spacer is preferably a divalent connecting group, which is represented by the following general formula:
  • U is a group which is coupled to the lipid and which is selected from the group consisting of -0-, -S-, -CO-, -COO-, -CONH-, -CSNH-, - (CN + H 2 ) -, -NH- and
  • W is a group which is coupled to the ligand and which is selected from the group consisting of -O-, -S-, -CO-, -OCO-, -NHCO-, -NHCS-, - (CN + H 2 ) -, -NH- and
  • V (a) is a branched or unbranched alkylene chain with 1 to 30 carbon atoms, the carbon atoms being partly by the groups -SS-, -CO-, -CH (OH) -, -NHCO-, -CONH-, cyclohexylene, phenylene, - COO-, -OCO-, -S0 2 -, -O- or -CH (CH 3 ) - can be replaced, and / or
  • the divalent connecting group include (a) a branched or unbranched alkylene chain with 1 to 30, preferably with 1 to 18 and particularly preferably with 1 to 12 carbon atoms, the carbon atoms being partly by the groups -SS-, -CO-, - CH (OH) -, -NHCO-, -CONH-, cyclohexylene, phenylene, -COO-, -OCO-, -S0 2 -, -0- or -CH (CH 3 ) - can be replaced, (b) one Polyethylene / propylene glycol chain with at least 3 ethylene / - propylene glycol units that can be branched, (c) a polyethylene / propylene amine chain with at least 3 ethylene / propylene amine units that can be branched, (d) an oligomeric or polymeric carbohydrate chain, (e) one Peptide chain, preferably an oligomeric or polymeric carbohydrate chain, (e) one
  • the materials that can be used to introduce a spacer into the tetraether lipids of the invention include commercially available products.
  • the products AEDP Product No. 22101ZZ
  • AMAS Product No. 22295ZZ
  • BMB Product No. 22331 ZZ
  • BMDB Product No. 22332ZZ
  • BMH Product No. 22330ZZ
  • BMOE Product No. 22323ZZ
  • BMPA Product No. . 22296ZZ
  • BMPH Product No. 22297ZZ
  • BMPS Product No. 22298ZZ
  • BM [PEO] 3 Product No. 22336ZZ
  • BM [PEO] 4 Product No.
  • the groups S 1 and S 2 can contain polyethylene glycol chains.
  • the materials that can be used to introduce polyethylene glycol chains into the tetraether lipids according to the invention include commercially available products. Polyethylene glycol chains are, for example, with average molar masses of 500 g / mol (average 11 ethylene glycol units), 1000 g / mol (average 23 ethylene glycol units, 2000 g / mol (average 45 ethylene glycol units), 3400 g / mol (average 77 ethylene glycol units) and 5000 g / mol (114 ethylene glycol units on average) These materials are preferably used according to the invention.
  • the circulation time of liposomes in the blood can be increased by incorporating lipids with polyethylene glycol chains (PEG chains), i.e. Liposomes containing such pegylated lipids have a longer lifespan in vivo than liposomes with unpegylated lipids.
  • PEG chains polyethylene glycol chains
  • the PEG chains preferably have molecular weights of 2000, 3400 or 5000 mass units.
  • the circulation time is highest when the pegylated lipid is well anchored in the liposome membrane. It is preferred that the bond between the lipid and the PEG part has a high stability.
  • the tetraether lipids are better anchored in the liposome membrane. This allows PEG chains, which are coupled to the TEL via stable bonds, to be anchored better in the liposome membrane than with conventional lipids. It has been shown that liposomes containing 5 mol% of the bis-pegylated tetraether lipid show a significantly lower release of carboxyfluorecein in the presence of serum than conventional liposomes (consisting of phosphatidylcholine), which indicates a significantly higher stability (see 3.3.2, Fig.8b).
  • pegylated TEL in serum, an increase in the release compared to the control is observed in liposomes in which conventional pegylated lipids are integrated.
  • pegylated TEL also represent a clear improvement of the conventional PEG system (see 3.3.2, Fig. 8b).
  • Bispegylated TEL derivatives are preferably provided for this.
  • the basic tetraether is linked via acid amide functions to two molecules of PEG, preferably the molecular weight 2000 (II).
  • Such a molecule shows better anchoring in the liposome membrane than e.g. the pegylated PE (phosphatidylethanolamine) lipids.
  • removal of such a bipolar lipid from the liposome membrane is made more difficult due to the second, voluminous, hydrophilic head group.
  • the link between the TEL derivative and the two PEG chains takes place via relatively stable acid amide bonds.
  • the circulation time of liposomes can be increased significantly.
  • the PEG chains can be replaced by poly (acrylic morpholine), poly (vinyl pyrrolidone) or gangliosides.
  • tetraether lipid-ligand conjugates according to the invention are outstandingly suitable for cell-specific targeting with the aid of long circulating liposomes.
  • pegylated tetraether lipids are particularly suitable for membrane anchoring of "large hydrophilic ligands, which greatly increase the tendency of the lipid-ligand conjugates to pass into the aqueous phase.
  • Preferred ligands are those selected from the group consisting of proteins, peptides, vitamins, carbohydrates and peptidomimetics.
  • the protein is preferably an antibody, a fragment of an antibody (Fab, F (ab) 2 or Fv), lectin or an apo-lipoprotein or a protein binding to a cell surface receptor.
  • the peptides are preferably peptides with 5 to 15 amino acids.
  • Other preferred peptides are EILDV, CDCRGDCFC, CNGRC and c (RGDfK).
  • the vitamins can be any essential or non-essential vitamin.
  • Preferred vitamins are vitamin B12 or folic acid.
  • peptidomimetics can also be used as ligands; be here preferably v ß 3 -specific diazepine derivatives or para-hydroxybenzoic acid amide derivatives used.
  • the ligand can contain, for example, a peptide sequence selected from CDCRGDCFC (cyclized via two disulfide bridges), EILDV, CNGRC (cyclized via a disulfide bridge), -c (RGDfK) - (cyclized via an amide bond), peptide sequences from the circumsporozoite protein and Transferrin peptide sequences.
  • the substituents S 1 and S 2 are the same at both ends of the tetraether lipid backbone. Based on natural tetraetheriipids, this enables synthesis without the interim use of protective groups.
  • the substituents S 1 and S 2 are different at both ends of the basic tetraether lipid structure.
  • Particularly preferred embodiments include those in which (a) S 1 comprises a cationic group and S 2 comprises a ligand, (b) S 1 comprises a neutral group and S 2 comprises a ligand and (c) S 1 comprises an anionic group and S 2 a Includes ligands.
  • This bifunctionality of the tetraether lipid derivatives according to the invention which enables the binding of a ligand possibly responsible for “targeting” outside and a substrate binding site inside a liposome, opens up undreamt-of therapeutic possibilities.
  • the tetraether lipid derivatives according to the invention can be in the form of salts.
  • Suitable counter cations include alkali metal ions such as sodium ions or potassium ions and ammonium ions.
  • Suitable counter anions include halide ions such as chloride ions, acetate ions and trifluoroacetate ions.
  • Other suitable anionic salts include fumerates, malonates, tartrates, citrates, succinates, palmoates, lactates and hydrogen phosphates.
  • the present invention also provides a composition containing two, three, four, five or more of the tetraether lipid derivatives according to the invention and optionally physiologically compatible additives.
  • the tetraether lipid derivatives according to the invention are preferably prepared from natural tetraether lipids, which e.g. can be isolated from archaebacteria.
  • pentacycles within the biphytanyl chains occur to a certain extent in the tetraetheriipids isolated from natural sources.
  • the extent of pentacyclization can be influenced by the cultivation temperature. Usually there are 0 to 8 pentacycles per tetraether backbone.
  • thermoplasmic acidophilum e.g. most lipid molecules at 1 to 5 pentatycles at a cultivation temperature of 39 ° C, while predominantly 3 to 6 pentacycles are observed at a cultivation temperature of 59 °.
  • the following table provides information on the distribution of pentacyclization in tetraetheriipids from Thermoplasma acidophilum at a cultivation temperature of 39 ° C or 59 ° C:
  • the tetraether lipid derivatives according to the invention can be obtained, for example, from the total lipid extract of archaebacteria, for example the total lipid extract of the archaebacterium Sulfolobus acidocaldarius, the total lipid extract of the archaebacterium Sulfolobus solfataricus or the total lipid extract of the archaebacterium Thermoplasma acidophilum.
  • Sulfolobus acidocaldarius was discovered in hot sulfur springs in Yellowstone National Park in 1972.
  • This archaebacterium grows between 60 and 90 ° C, with a temperature optimum of 78 ° C.
  • the limiting pH values for growth are between 3.0 and 3.5, with the optimum at 3.3.
  • Sulfolobus acidocaldarius grows optimally under microaerophilic conditions, ie only a low oxygen concentration in the medium is tolerated; too high 0 2 concentrations have a toxic effect.
  • Sulfolobus acidocaldarius is optional chemolithotrophic and therefore obtains its energy in natural habitats from the oxidation of elemental sulfur. Since sulfolobes can also grow organothrophically, this form of nutrition is used for cultivation.
  • Sulfolobus acidocaldarius contains tetraether lipids in its cell membrane, which only occur in archaebacteria. The tetraether lipids impart special properties such as pH and temperature stability of the cells, which make the life of the bacteria possible under these conditions.
  • the starting compounds used for the preparation of the tetraether lipid derivatives according to the invention can be obtained from the total lipid extract of archaebacteria using conventional methods. Such methods are e.g. in WO-A-97/31927 and in WO-A-99/10337.
  • the starting compounds which are used to prepare the tetraether lipid derivatives according to the invention can of course also be prepared synthetically. Appropriate methods are described in T. Eguchi et al., J. Org. Chem. 1998, 63, 2689-2698; K. Arakawa et al., J. Org. Chem. 1998, 63, 4741-4745; and in T. Eguchi et al., Chem. Eur. J. 2000, 6, 3351-3358.
  • the liposomes and lipid agglomerates according to the invention contain one or more of the tetraether lipid derivatives described above.
  • the liposomes or lipid agglomerates can comprise one or more layers, each containing one or more of these tetraether lipid derivatives.
  • lipid intended for the preparation of the liposomes is first dissolved in an organic solvent and a lipid film is formed by evaporation.
  • the lipid film will be good dried to remove all solvent residues.
  • the lipids of this film are then resuspended in a suitable buffer system.
  • physiological saline, pH 7.4 is suitable, but other buffer systems (for example Mcllvaine buffer), or unbuffered solutions, such as, for example, unbuffered potassium or sodium chloride solutions, can also be used.
  • vesicles By shaking by hand, large multilamellar vesicles with a size distribution in the ⁇ m range can be formed.
  • the formation of these vesicles can be facilitated by using two glass spheres and / or an ultrasound bath with low sound intensity.
  • Liposomes with a diameter of around 500 nm are formed.
  • the liposomes can be centrifuged in a 3200 Eppendorf centrifuge for 10 minutes in order to remove non-liposomal material. Intact, closed vesicles remain in the supernatant.
  • Liposomes can be further produced by detergent solubilization with subsequent detergent dialysis.
  • a lipid film is first formed as described above. This is suspended in a detergent-containing buffer system (examples of dialysable detergents: octyl- ⁇ -D-glucopyranoside or octyl- ⁇ -D-thioglucopyranoside).
  • the molar ratio (TEL derivative: detergent) for the detergents mentioned should be between 0.05 and 0.3 and the amount of buffer should be calculated in such a way that a liposome dispersion with a maximum of 15-20 mg lipid per ml buffer subsequently results.
  • Mixing micelles from detergent and TEL derivative is formed by shaking by hand.
  • the suspension of the mixed micelles is now in dialysis tubes, e.g. transferred into a Lipoprep® dialysis cell or into a Mini-Lipoprep® dialysis cell (Diachema AG, Langnau, Switzerland) and dialyzed at RT for 24 hours.
  • the mixed micelles form liposomes with a diameter of approximately 400 nm.
  • the liposome preparation can be centrifuged in a 3200 Eppendorf centrifuge for 10 minutes to remove non-liposomal material. Intact, closed vesicles remain in the supernatant.
  • a preferred protocol for liposome preparation by detergent dialysis, in which detergent sodium cholate is used, is described in the examples.
  • Liposomes which contain the TEL derivatives according to the invention (at least 10% TEL content in the lipid layer) or which are made up of 100% TEL derivatives have proven to be exceptionally stable. It has been shown that the shelf life of liposomes containing TEL components increases many times over that of conventional liposomes.
  • tetraether lipid derivatives are suitable as an additive for liposomes, the stability of which and thus drug release should be controlled in a targeted manner.
  • TEL derivative liposomes are therefore the method of choice.
  • octreotide As an example, it was shown that the oral intake of octreotide is greatly increased by the liposomal formulation with TEL-containing liposomes (see 4.4.3.2, Fig. 18).
  • the production of liposomes which contain conventional double-layer-forming phospholipids in addition to a proportion of TEL derivative has also proven to be advantageous.
  • the liposome membranes become more rigid and less permeable than the conventional liposomes.
  • the production Mixed liposomes are analogous to the production of pure TEL derivative liposomes.
  • Preferred bilayer forming phospholipids used in the present invention include bilayer forming cationic, neutral or anionic lipids.
  • the liposomes and lipid agglomerates according to the invention can contain the cationic lipid DOTAP® (Röche Diagnostics, Germany) and / or DOSPER® (Röche Diagnostics, Germany) and / or DC-Chol®.
  • the liposomes and lipid agglomerates according to the invention contain the neutral lipids phosphatidylcholine and / or phosphatidylethanolamine and / or phosphatidylglycerol and / or phosphatidylserine and / or phosphatidic acid and / or cholesterol and / or sphingomyelin.
  • the weight ratio of tetraether lipid derivative to further lipids in the liposomes and lipid agglomerates according to the invention is preferably 5: 1 to 1: 100.
  • the liposomes and lipid agglomerates according to the invention can furthermore contain nucleic acid molecules and / or compounds analogous to nucleic acid molecules (such as phosphorothioate) and optionally polycations.
  • Preferred polycations include polyethyleneimine, poly-lysine and protamine.
  • the liposomes according to the invention including the mixed liposomes and the lipid agglomerates including the mixed agglomerates can serve as transport vehicles for nucleic acids and / or cosmetic and / or pharmaceutical active substances.
  • Active pharmaceutical ingredients can be, for example, antibiotics, cytostatics or growth factors.
  • the active pharmaceutical ingredients can be produced synthetically or recombinantly. They can have further modifications, such as glycosylation, acetylation or amidation.
  • “Mixed liposomes” and “mixed-lipid agglomerates” additionally include conventional phospholipids.
  • the liposomes and lipid agglomerates according to the invention also enable targeted gene transfer or targeted drug delivery.
  • nucleic acids for example DNA or RNA sequences, which contain genes or gene fragments and are present in linear form or in the form of circularly closed vectors which may contain further genetic material, or else pharmaceutical or cosmetic active substances in pure form TEL liposomes or mixed liposomes, pure lipid agglomerates and mixed agglomerates are packaged and added to the target cells in vitro or in vivo. If the liposome membrane or agglomerate surface also z.
  • the contact between the antigen on the target cell and the antibody in the membrane of the liposome or agglomerate surface according to the invention results in a contact between the liposome or agglomerate surface and Target cell promoted.
  • the other ligands mentioned above which can react with substances on the target cell surface.
  • the pharmaceutical composition according to the invention contains the liposomes or lipid agglomerates described above and a physiologically tolerable diluent.
  • the present invention also provides an in vitro transfection kit which contains the liposomes and / or lipid agglomerates according to the invention and suitable buffers.
  • the liposomes or lipid agglomerates according to the invention can be used to produce a medicament for gene therapy in mammals.
  • the liposomes or lipid agglomerates according to the invention can also be used for the transfection of eukaryotic cells in vitro.
  • Another embodiment of the invention relates to the use of the liposomes or lipid agglomerates according to the invention for packaging active substances for oral, enteral, parenteral, intravenous, intramuscular, intra-articular, topical, subcutaneous, pulmonary or intraperitoneal application.
  • the TEL derivatives according to the invention are used in pure form or as a constituent of pure or mixed liposol or lipid agglomerates as the basis for the manufacture of medical ointments or skin creams.
  • tetraether lipid derivatives and compositions according to the invention can be used for coating surfaces, in particular metal or plastic surfaces.
  • Tetraether lipids can be covalently coupled to surfaces (e.g. stents, implants) as a monomolecular layer. Examples of such processes are described in WO-A-97/31927.
  • hydrophobic active substances can be enclosed like in a membrane. Active ingredients can also be coupled to the hydrophilic head groups.
  • the surfaces loaded with active substances according to these possibilities permit the slow and continuous release of active substance over longer periods of time. In this way, for example, the compatibility of coated stents or implants can be increased.
  • surfaces to be coated examples include oxidic surfaces (e.g. oxide layers on titanium), ceramic surfaces, semiconductor surfaces, glass surfaces, glassy carbon surfaces, cellulose foils (the coupling takes place via cyanuric chloride activation and coupling), gold surfaces (the coupling takes place via SH groups (either electrochemically or eg via iminothiolan (Mitsunobu reaction)) and polymer surfaces such as polyurethane surfaces, Teflon surfaces, polystyrene surfaces, polyacrylic resin surfaces (coupling depending on the reactive groups on the polymer surface).
  • oxidic surfaces e.g. oxide layers on titanium
  • ceramic surfaces semiconductor surfaces
  • glass surfaces glassy carbon surfaces
  • cellulose foils the coupling takes place via cyanuric chloride activation and coupling
  • gold surfaces the coupling takes place via SH groups (either electrochemically or eg via iminothiolan (Mitsunobu reaction))
  • polymer surfaces such as polyurethane surfaces, Teflon surfaces, polystyrene
  • Figure 1 shows (a) the density gradient centrifugation of liposomes from phosphatidylcholine and compound II and (b) the serum stability of liposomes from phosphatidylcholine and compound II.
  • Figure 2 shows the particle size determination of the mixed liposomes from compound VI and phosphatidylcholine.
  • Figure 3 shows the stability of liposomes containing compounds V and VI in the presence of detergents and serum.
  • Figure 4 shows the plasma level of octreotide after oral administration of liposomal formulation VI or after administration of the free substance.
  • Figure 5 shows the transfection of CHO cells with liposomes containing compound X.
  • Figure 6 shows the specific transfection efficiency of compound IX compared to AF1.
  • DGTE Glycerol dialkyl glycerol tetraether: approx. 10% of the total lipid content
  • GCTE Glycerol dialkyl calditol tetraether: approx. 40% of the total lipid content
  • the GCTE consists of two different lipids, which differ in the head group area (see following figure. ). Compound 3 can be converted to DGTE (2) by glycol cleavage with sodium metaperiodate.
  • the macro cycles of the tetraethers shown contain 0-8 pentacycles per macro cycle (four are shown in the following figure). In the other figures, the macro cycles are only symbolized by a box.
  • the silica gel is swollen in chloroform / diethyl ether (9: 1).
  • the starting compounds used in the following examples are obtained from the tetraetheriipids obtained using known methods. Such methods are e.g. in WO-A-97/31927 and in WO-A-99/10337.
  • the mass spectrum shows a set of equidistant lines with a spacing of 44 mass units (ethylene oxide unit).
  • the distribution of the heights of these lines corresponds to a Gaussian distribution.
  • IR spectroscopy (NaCI): v 1672, 1524 cnr ⁇ 1 (amide l + ll), 1112 (ether).
  • the mass spectrum shows a set of equidistant lines with a spacing of 44 mass units (ethylene oxide unit).
  • the distribution of the heights of these lines corresponds to a Gaussian distribution.
  • IR Spectroscopy (KBr): v 2923cm -1 (alkyl), 1733 (carboxyl), 1672 (amide), 1464, 1360 (alkyl), 1113 (ether).
  • the compound II and phosphatidylcholine (from protein) are mixed in a molar ratio of 1: 0.02 and dissolved in 2 ml of a mixture of chloroform: methanol (1: 1) (v / v).
  • the total lipid content of the batches is 9800 nmol.
  • the chloroform-methanol mixture of the dissolved lipids is removed on a rotary evaporator and the lipid film is dried for 15 minutes at 40 ° C. and 300 mbar and then for 15 minutes at 40 ° C. and 20 mbar.
  • the dry lipid film is taken up in 1 ml of bidistilled water and an aqueous suspension of multilamellar liposomes is prepared by shaking overnight at room temperature (Heidolph Unimax 1010 laboratory shaker).
  • the multilamellar liposomes are made using a hand extruder (Avanti Polar Lipids) by twenty extrusions through a 10Onm polycarbonate membrane, large unilamellar liposomes.
  • the aqueous suspension of the large unilamellar liposomes is lyophilized overnight.
  • the lyophilisate obtained is taken up in 50 ⁇ l of double-distilled water, incubated for one hour at room temperature, made up to 1 ml by adding 950 ⁇ l of double-distilled water and extruded again 20 times through a 100 nm polycarbonate membrane by means of the hand extruder.
  • a gradient mixer 7.5 ml of bidistilled water and 7.5 ml of 30% (w / w) sucrose in bidistilled water in a 16 x 96 mm ultra-clear centrifuge tube (Beckman Coulter) is used to make a continuous sucrose density gradient (0 to 30% (w / w) sucrose). Then 0.5 ml of the aqueous liposome suspension to be analyzed is mixed with 0.5 ml of 66% (w / w) sucrose in bidistilled water and layered under the sucrose density gradient.
  • the centrifuge tube with the liposome suspension layered under the sucrose density gradient is centrifuged in a Beckman Coulter Avanti J-30I high performance centrifuge (Beckman Coulter JS-24.15 rotor) for 16 hours at 20 ° C. and 100000 g. After centrifugation, the density gradient is fractionated (fraction volume: 0.7 ml). The individual fractions are collected in 1.5 ml reaction vessels. Fraction 1 was always the fraction with the highest sucrose content and fraction 22 the fraction with the lowest sucrose content. The phospholipid content of the individual fractions is determined by an enzymatic choline assay (Phospholipides Enzymatiques PAP 150-Kits; Biomerieux). Then all fractions are lyophilized overnight.
  • the lyophilisates obtained are taken up in 1 ml each of chloroform: methanol (1: 1) (v / v), sonicated for one hour in an ultrasound bath and then in a Sigma 4K15 centrifuge (Sigma 12130 -H rotor) centrifuged. The supernatants are completely removed in each case, transferred to 1.5 ml reaction vessels and dried in vacuo at 40 ° C. The dry centrifugation supernatants are taken up in 30 ⁇ l chloroform: methanol (1: 1) (v / v) and placed on silica gel 60 F 25 HPLC plates (Fa. Merck) applied.
  • the compound II and the lipid phosphatidylcholine (from protein) are mixed in the molar ratios 1: 0.02 and 1: 0.05 and dissolved in 2 ml of a mixture of chloroform: methanol (1: 1) (v / v).
  • the total lipid content of the batches is 5000 nmol.
  • the chloroform-methanol mixture of the dissolved lipids is removed on a rotary evaporator and the lipid film is dried at 40 ° C. and 300 mbar for 15 minutes and then at 40 ° C. and 20 mbar for 15 minutes.
  • the dry lipid film is taken up in 1 ml of double-distilled water and an aqueous suspension of multilamellar liposomes is prepared by shaking overnight at room temperature (Heidolph Unimax 1010 laboratory shaker).
  • the multilamellar liposomes are produced using a hand extruder (Avanti Polar Lipids) by 20 extrusions through a 100 nm polycarbonate membrane and large unilamellar liposomes.
  • the aqueous suspension of the large unilamellar liposomes is lyophilized overnight.
  • the lyophilisate obtained is taken up in 100 ⁇ l of a 2.5% solution of carboxyfluorescein in KRB buffer, incubated for one hour at room temperature and made up to 1 ml by adding 950 ⁇ l KRB buffer (KRB buffer: 114 mM sodium chloride, 5 mM potassium chloride , 1.65 mM disodium hydrogen phosphate, 0.3 mM sodium dihydrogen phosphate, 20 mM sodium hydrogen carbonate, 10 mM HEPES, 25 mM gluco- se).
  • the lipid suspension obtained is transferred into 100 nm liposomes by extrusion through a 100 nm polycarbonate membrane 20 times (hand extruder; Avanti Polar Lipids).
  • the separation of carboxyfluorescein not included is carried out by size exclusion chromatography (SEPHADEX G-75; gel bed 1 cm in diameter, 25 cm in length; running buffer: KRB).
  • the liposomes with the incorporated carboxyfluorescein are used in serum stability studies.
  • the measuring principle and implementation of the serum stability tests are described in 4.4.2.2. "Stability studies in biological environments" described.
  • the serum stability of the liposomes containing compound II is shown in Figure 1.b.
  • liposomes containing 5 mol% of compound II show a significantly lower release of carboxyfluorescein than conventional liposomes (consisting of phosphatidylcholine), which indicates a significantly higher stability.
  • compound II In contrast to compound II, an increase in release compared to the control is observed in liposomes in which conventional pegylated lipids are integrated (Nikolova and Jones, Biochim. Biophys. Acta 1996, 1304, 120-128.). With regard to the release properties, compound II thus represents a clear improvement of the conventional PEG system.
  • the aqueous phase is extracted 10 times with chloroform / methanol (2: 1).
  • the combined organic phases are dried over sodium sulfate and the solvent is removed in vacuo (crude yield: 98.7 mg).
  • the residue is purified twice using column chromatography on silica gel (15 g of silica gel 60 (0.040-0.063 mm; eluent: chloroform / methanol / water / 37% ammonia solution in water (60: 34: 5.5: 0.5)).
  • anhydrous tetrahydrofuran 150 ⁇ l of anhydrous tetrahydrofuran is placed under a nitrogen atmosphere. At 0 to 5 ° C (ice bath) 6.20 ⁇ l (68.2 ⁇ mol) phosphoryl chloride and then 10.4 ⁇ l (74.9 ⁇ mol) triethylamine are slowly added via a syringe. With good stirring, 44.3 mg (34.0 ⁇ mol) of anhydrous DGTE are slowly added dropwise at 0 ° C. within 15 min.
  • the reaction mixture is mixed with a solution of 4.50 ⁇ l (74.9 ⁇ mol) anhydrous ethanolamine, 37.7 ⁇ l (270 ⁇ mol) triethylamine and 88.6 ⁇ l anhydrous tetrahydrofuran.
  • the aqueous phase is extracted 10 times with chloroform / methanol (2: 1).
  • the combined organic phases are dried over sodium sulfate and the solvent is removed in vacuo.
  • the oily residue is purified by column chromatography over silica gel (15 g silica gel (0.04-0.063 mm).
  • the mobile phase is chloroform / methanol (3: 1) and then, after the DGTE has been completely eluted, chloroform / methanol / water / 37 % Ammonia solution in water (60: 34: 5.5: 0.5) and the solid is purified by crystallization from chloroform / methanol (2: 1). Yield: 11 mg (20%), colorless solid.
  • the bromoethyl dichlorophosphate reagent is prepared according to the following literature: Eibl et al., Chem. Phys. Lipids 1978, 22, 1-8. 1st stage: synthesis of DGTE-PBr
  • the phases are separated and the aqueous phase is extracted 5 times with 25 ml of chloroform / methanol (2: 1).
  • the combined organic phases are then dried over sodium sulfate and the solvent is removed in vacuo.
  • the brown oil is dried in an oil pump vacuum.
  • the purification is carried out using two column chromatographs:
  • DGTE-PBr 104 mg (62.3 ⁇ mol) DGTE-PBr are dissolved in 9 ml chloroform, 7.5 ml 2-propanol and 2.5 ml water and mixed with 6.5 ml of a 31 - 35% trimethylamine solution in water. The mixture is stirred for 4 days with the exclusion of light at room temperature. The solution is then mixed with 20 ml of a chloroform / methanol mixture (2: 1) and then with 5 ml of a semi-saturated sodium chloride solution. The phases are the separated and the aqueous phase is extracted 5 times with 10 ml of chloroform / methanol (2: 1).
  • the compound V and the lipid phosphatidylcholine (from protein) are mixed in different ratios (see 4.4.1.2.) And dissolved in 1 ml of a mixture of chloroform and methanol (2: 1). The total lipid content of the batches is 120 nmol in each case. After adding 480 nmol sodium cholate as detergent, the mixture is evaporated on a rotary evaporator, taken up in 1 ml of ethanol, evaporated again and dried for 30 min at 50 ° C. and 20 mbar. The system compound V / phosphatidylcholine forms a homogeneous, transparent film in all examined mixing ratios.
  • the lipid detergent film is taken up in 400 ⁇ l dialysis buffer (10 mM HEPES, pH 7.4, 150 mM NaCI), transferred into a dialysis capsule (Carl Roth GmbH, Düsseldorf, Germany) and provided with a dialysis membrane (exclusion limit 10 kDa, Dianorm GmbH, Kunststoff).
  • the lipid / detergent mixture is dialyzed for 36 h against 1000 times the volume of dialysis buffer. During this time, the dialysis buffer is replaced three times.
  • composition of the liposomes is examined using thin layer chromatography.
  • 20 ⁇ l of the liposome solution are mixed with 80 ⁇ l chloroform: methanol (2: 1), shaken for 5 min and centrifuged at 14,000 g for 5 min.
  • the upper, aqueous phase is removed and discarded.
  • the organic phase is evaporated under nitrogen, taken up in 20 ⁇ l chloroform: methanol (2: 1) and applied to an HPTLC thin-layer plate (Merck, Darmstadt, Germany).
  • the plate After developing the HPTLC in the eluent chloroform: methanol: water: 32% ammonia solution in water 60: 34: 5.5: 0.5, the plate is briefly immersed in the staining solution (3% (w / v) copper acetate in 8% (v / v ) Phosphoric acid) immersed and heated at 180 ° C for 20 min.
  • the lipids are identified by comparison with lipid standards.
  • the particle size is determined by dynamic light scattering using a party e-sizing system (380 ZLS, Nicomp Inc., Santa Barbara, CA, U.S.A.) in a sample volume of 150 ⁇ l. The particle size is weighted based on a Gaussian distribution.
  • the composition of the liposomes was checked by thin layer chromatography and corresponded to the composition in which the two lipids were used, which indicates that there was no loss of any of the components during manufacture.
  • the size of the liposomes was dependent on the lipid Composition and increased almost linearly with the content of compound V (in mol%).
  • Fig. 2 shows the results of particle size determination. The results show that compound liposomes can be prepared from compound V and the phosphatidylcholine by detergent dialysis in a wide concentration range (up to 100% compound V).
  • Compound V or compound VI and the lipid phosphatidylcholine (from protein) are mixed in different ratios (0-75 layer% compound V / Vl) and dissolved in 1 ml of a mixture of chloroform and methanol (2: 1). The total lipid content of the batches is between 100 and 1000 nmol. The mixture is evaporated on a rotary evaporator and dried for 15 min at 50 ° C. and 20 mbar.
  • the lipid film obtained is taken up with 1 ml of a 2.5% solution of carboxyfluorescein solution in KRB buffer and, after addition of 10 Teflon balls, shaken overnight at 250 rpm (KRB buffer: 114 mM NaCl, 5 mM KCI, 1.65 mM Na 2 HP0, 0.3 mM NaH 2 P0, 20 mM NaHCO 3 , 10 mM HEPES, 25 mM glucose).
  • the lipid suspension is transferred into 100 nm liposomes by extrusion (hand extruder; Avanti Polar Lipids).
  • the separation of not included carboxyfluorescein is carried out by size exclusion chromatography (SEPHADEX G-75; gel bed 1 cm0, 25 cm length; running buffer: KRB).
  • Liposomes can be produced in the range of 0-75 layer% compound V or VI using this method. In all preparations, liposomes are obtained which have incorporated carboxyfluorescein, which indicates a closed structure of the liposomes.
  • DGTE-PE chloroform: methanol: water: 32% ammonia solution in water (60: 34: 5.5: 0.5)
  • DGTE-PC chloroform: methanol: water: 32% ammonia solution in water (35: 25: 3: 2.5)
  • the analysis of the lipid composition shows that the composition of the liposomes in the range of 0 - 75 layer% V or VI corresponds to their initial weight at the beginning of the preparation, whereby the complete incorporation of compounds V and VI into the lipid matrix is verified.
  • the liposomes with incorporated carboxyfluorescein (CF) are used in stability studies. The measurement is based on the increase in the fluorescence intensity of CF when released from lipsomes.
  • the stability of the liposome in the presence of detergents is crucial for possible use as an oral drug delivery system. Stability in the serum is a prerequisite for using the liposome as an intravenous drug.
  • ⁇ l of the liposome sample is mixed with 75 ⁇ l of a 0.4% sodium cholate solution in KRB.
  • the change in fluorescence is monitored over a period of 50 seconds (excitation 485 nm; emission 538 nm).
  • the release of CF (in%) is calculated from the comparison of the measured value with the 0% sample (liposomes before the measurement) and the 100% sample (liposomes which were previously incubated for 10 minutes with a 4% sodium cholate solution).
  • the measurement is carried out as a four-fold determination in 96-well microtiter plates according to the time-resolved mode (Fluoroskan Ascent, Thermo-Labsystems).
  • 60 ⁇ l liposomes are mixed with 240 ⁇ l fetal calf serum and incubated at 37 ° C. for 16 h. The fluorescence intensity is measured every hour using the parameters described above.
  • 60 ⁇ l of a liposome solution which is mixed with 240 ⁇ l KRB buffer and carried under the same conditions, serve as a 0% sample.
  • the 100% sample consists of liposomes that were previously digested by adding a 4% sodium cholate solution in KRB.
  • the stability of the liposomes containing compounds V and VI is shown in Fig. 3.
  • the liposomes with V and VI show a significantly lower release of carboxyfluorescein than conventional liposomes (consisting of PC), which indicates a significantly higher Stability indicates.
  • the stabilization depends on the concentration and is more pronounced with compound V than with compound VI.
  • the data on the serum stability can be expected that the release of the liposomes can be adjusted continuously by changing the content of V or VI and thus enable a controlled release system for drug release.
  • the somatostatin-like peptide octreotide (size 8 amino acids) serves as a model compound for testing liposomes from VI as an oral drug delivery system.
  • To produce a lipid film 10 ⁇ mol of compound VI and 20 ⁇ mol of phosphatidylcholine are dissolved in 1 ml of chloroform: methanol (2: 1) and evaporated on a rotary evaporator. The film is dried for 30 min at 50 ° C. and 20 mbar and then after adding 2 ml bidistilled. Shaken water and 10 Teflon balls overnight at 250 rpm.
  • the resulting suspension of multilamellar vesicles is sonicated using a sonotrode for 60 min (Branson Sonifier; 70% amplitude) and thus transferred into small unilammelar vesicles (SUV).
  • the SUVs have an average size of 70 nm (for particle size determination see 4.4.1.2.).
  • 15 ⁇ mol lipid are transferred to a reaction vessel, frozen at -70 ° C. and lyophilized.
  • 2 mg octreotide are dissolved in 20 ⁇ l PBS and the lyophilisate is hydrated with this solution. After complete hydration, the liposome suspension is diluted to 1 ml.
  • the liposomes are centrifuged at 14,000 g for 20 min and the supernatant solution is removed. The washing step is repeated twice and the pellet obtained is resuspended in 500 ⁇ l PBS.
  • This method allows octreotide to be incorporated into PC and compound VI liposomes with an efficiency of approximately 10%.
  • the octreotide is quantified by HPLC and comparison with a calibration series of free octreotide: quantification of the octreotide by HPLC: eluent: acetonitrile / phosphate buffer solution (1: 1) Phosphate buffer solution:
  • octreotide (as a liposomal formulation or free) is administered orally into the stomach using a gavage.
  • 200 ⁇ l of retroorbital blood is withdrawn from the rats after anesthesia.
  • the plasma samples are frozen and the octreotide is then quantified by a radio-immunoassay (MDS-Pharma).
  • Fig. 4 shows the plasma concentrations of octreotide after oral administration of the liposomal formulation or the free substance. The values are mean values from 3 animals each.
  • the liposomal formulation of octreotide with the compound VI greatly increases the oral absorption of octreotide: the AUC ("Area under the Curve) of octreotide is doubled, while the clearance kinetics of octreotide remain unaffected. This is a clear indication of the Suitability of compound VI liposomes as an oral drug delivery system.
  • the solvent is evaporated and the remaining yellow oil is chromatographed on silica gel (about 130 g of silica gel 60, 0.063-0.200 mm, eluent: ethyl acetate / methanol (3: 2 +0.5% glacial acetic acid)).
  • the solvent of the fractions collected is removed in vacuo and the residue is dissolved in ethyl acetate.
  • the organic phase is washed once with 1 M sodium hydroxide solution and once with water and dried over sodium sulfate. The solvent is in. Vacuum removed and the residue dried.
  • the BOC-AF7 is dissolved in chloroform and stirred for 15 minutes with trifluoroacetic acid (TFA) to remove the BOC protective groups.
  • TFA trifluoroacetic acid
  • the crude product is purified by chromatography twice over silica gel (both columns each about 5 g silica gel 60, 0.040-0.063 mm).
  • t? -Hexane / ethyl acetate (1: 1) is used as the eluent
  • the second is carried out with pure ethyl acetate.
  • BOC-AF7 (XI) are stirred with 0.2 ml chloroform and 0.1 ml trifluoroacetic acid (TFA) for 15 min in order to remove the BOC protective groups.
  • the liposomes are prepared by detergent dialysis according to the method described in 4.4.1.1. described method.
  • the lipids phosphatidylcholine (from protein), cholesterol (from wool fat) and dioleylphosphatidylethanolamine (synthetic) are used in various ratios.
  • the lipids are reacted with a maximum of 1 mM total lipid with ten times the molar amount of sodium cholate.
  • CHO cells Chinese hamster ovary cells
  • CHO cells Chinese hamster ovary cells
  • the cultivation takes place in Iscoves Modified Dulbeccos medium with 10% fetal calf serum, 2 mM glutamine .
  • Liposomes containing compound X are introduced in various amounts of 0.25 -6.0 nmol compound X and filled up to a volume of 60 ⁇ l with 10 mM HEPES, pH 7.4, 150 mM sodium chloride.
  • the batches are mixed with 70 ⁇ l Opti-MEM (Life Technologies, Düsseldorf, Germany) and incubated for 30 min at room temperature. 0.15 ⁇ g plasmid DNA are added to 70 ⁇ l Opti-MEM for each batch and incubated for 15 min at room temperature.
  • the CHO cells are washed twice with PBS, 150 ⁇ l of the transfection mixture are added and the mixture is incubated at 37 ° C. for 5 h. The transfection solution is then removed, replaced by 400 ⁇ l of culture medium and incubated for a further 24 h.
  • ⁇ -galactosidase or the green fluorescent protein (GFP) pCMVß or pEGFP-N2; Clontech Inc., Palo Alto, CA, USA.
  • GFP green fluorescent protein
  • the ⁇ -galactosidase activity expressed in the cells is determined.
  • the cells in the 48-well plates are washed three times with PBS, with 150 ⁇ l bidist. Water was added, frozen at -70 ° C. and thawed again and then centrifuged at 3000 g for 30 min.
  • Compound X can be used to produce the three co-lipids phosphatidylcholine (PC), cholesterol (Chol) and dioleylphosphatidylethanolamine (PE) in different ratios.
  • PC phosphatidylcholine
  • Chol cholesterol
  • PE dioleylphosphatidylethanolamine
  • Liposomes with the composition X: PC: PE: Chol (1: A: B: C with A, B, C between 0.5 and 2) can be represented. All liposomes show more or less strong transfection properties.
  • liposomes with the composition X: PC: PE: Chol (1: 1: 1: 1) (mol%) will be presented.
  • the transfection efficiency shows a strong dependence on the ratio of compound X to the DNA content of the transfection approach with an optimum at 3 nmol X / ⁇ g DNA.
  • Liposomes from IX basically show the same transfection behavior as liposomes from X.
  • the transfection optimum is around 7.5 nmol / ⁇ g DNA.
  • compound IX represents a significant improvement.
  • compound IX is 4-5 times more efficient than AF1.
  • Compound IX shows no loss of transection efficiency in the presence of serum, while a drastic decrease in the efficiency of serum addition can be observed in AF1.

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Abstract

L'invention concerne de nouveaux dérivés de tétraétherlipides offrant une stabilité améliorée dans la préparation de liposomes et d'agglomérats lipidiques d'utilisation simple et fiable. La présente invention porte également sur des liposomes et des agglomérats lipidiques présentant une plus grande longévité in vivo.
EP01985932A 2000-12-28 2001-12-28 Derives de tetraetherlipides, liposomes contenant des derives de tetraetherlipides et agglomerats lipidiques ainsi que leur utilisation Withdrawn EP1347964A2 (fr)

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DE102004033667A1 (de) * 2004-07-01 2006-02-02 Institut für Bioprozess- und Analysenmesstechnik e.V. Kompositmaterialien mit Tetraetherlipiden und deren Herstellung
DE102012216378B4 (de) * 2012-09-14 2014-05-15 Institut für Bioprozess- und Analysenmesstechnik e.V. Immobilisierungsmatrix mit Tetraetherlipidschicht, Verfahren zu deren Herstellung und Biosensorchip umfassend diese Immobilisierungsmatrix
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DE19607722A1 (de) * 1996-02-29 1997-09-04 Freisleben H J Dr Tetraetherlipide und diese enthaltende Liposomen sowie deren Verwendung
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