WO2002053554A2

WO2002053554A2 - Tetraether lipid derivatives and liposomes containing tetraether lipid derivatives and lipid agglomerates and the use thereof

Info

Publication number: WO2002053554A2
Application number: PCT/EP2001/015356
Authority: WO
Inventors: Christine KÜHL; Bernhard Tewes; Martin Hagen; Felix Gropp; Ralf Littger; Uwe Marx
Original assignee: Bernina Biosystems Gmbh
Priority date: 2000-12-28
Filing date: 2001-12-28
Publication date: 2002-07-11
Also published as: WO2002053554A3; DE10065561A1; EP1347964A2

Abstract

The invention relates to novel tetraether lipid derivatives which can be used for producing liposomes and lipid agglomerates with improved stability which are simple and reliable. The invention also relates to liposomes and lipid agglomerates with an increased lifespan in vivo.

Description

Liposomes and lipid agglomerates containing tetraether lipid derivatives and tetraether lipid derivatives and their use

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.

Scientific research has a great need for methods for the transfection of cells in cell culture and of multicellular organisms with nucleic acids. The conventional methods such as electroporation, DEAE- and "calcium phosphate" -assisted transfection, microinjection or ballistic methods have the disadvantage that they often only achieve low transfection efficiencies, the cell exaggeration rates are very low and / or that they cannot be carried out on multicellular cells. Viral and retrovirus transfection systems are more efficient, but involve their own risks, such as an increased immune response or uncontrolled integration into the target genome. The transfection of non-viral nucleic acids with the help of liposomes, also called lipofection, therefore represents a successful and already frequently used alternative to the methods described above.

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. In addition to the use mentioned above as a lipofection agent, ie as a transport vehicle for nucleic acid ren, liposomes are widely used as transport vehicles for therapeutic agents. For this function, which has become known under the keyword “drug delivery”, 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 , For example, the cosmetics industry produces liposome-containing skin creams that transport active ingredients into the epidermis and lower cell layers. In this context, 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. By varying the 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.

One of the major disadvantages of today's liposomes is their poor stability. 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 α-tocophero, but the stability thus improved is still insufficient for many purposes. In addition, 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. In addition to the disadvantages already mentioned, 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. Furthermore, 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.

However, there is still a need for lipids. which enable the production of liposomes which are improved with regard to their in vivo stability or storage stability. There is also a need for 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. This object is achieved according to the invention by providing tetraether lipid derivatives (hereinafter also abbreviated as "TEL derivatives"), represented by one of the following general formulas (1) to (6):

where S ¹ and S ² , which may be the same or different, are represented by the following formula:

-A- (X ¹ ) _r -B- (X ² ) _q -Y

in which mean:

A -CONR ¹ -, -COO-, -CH ₂ 0-, -CH ₂ OCO-, -CH ₂ OCOO-, -CH ₂ NR ¹ CO-, -CH ₂ OCONR ¹ -, -CH ₂ NR ¹ COO- , -CON [(CH ₂ ) _m Y] -, -CH ₂ OCO (CH ₂ ) _m O- -CONH (CH ₂ ) _m NHCO (CH ₂ ) _m O-, -CH ₂ COO-, -CH ₂ 0 (P0 ₂ R ¹ ) O- or -CH ₂ S-,

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 -,

B - [(CH ₂ ) _m O] _n -, - [O (CH ₂ ) _m ] _n -, - [NR ¹ COCHN (R ¹ ) ₂ ] ₀ - or - (NR ⁶ ) _P -,

Y -H, -R ² , -NR R ³ , -N ⁺ R ² R ³ R ⁴ , -N [(CH ₂ ) _m Y] ₂ , -NR ² (CH ₂ ) _m Y, -OR ⁵ , - COR ⁷ ,

-NHC (NH) NH ₂ , -NHCOY or -SR ⁸ , where Y can have different meanings within a group,

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 ,

R ² to R ⁵ ,

R ⁷ and R ⁸ independently of one another R ¹ or - (C = N ⁺ H ₂ ) NH ₂ ,

m 1 to 5, where m can assume different values within a group, n 0 to 150, o 0 to 10, p 0 or 1, q 0 to 5 and r 0 or 1,

where in each case 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, and modifications thereof formed by the formation of pentacycles in the basic tetraether structure,

and salts of these compounds,

with the proviso that when the tetraether lipid derivative is represented by the general formula (1), the following compounds are excluded:

A = -CONH-, B = - (NR ⁶ ) _P - with p = 1 and R ⁶ = -H or a branched or unbranched unsubstituted alkyl, alkenyl, aralkyl or aryl group with 1 to 12 carbon atoms;

A = -CONH-, n = o = p = q = 0 and Y = -NR ² R ³ or -N ⁺ R ² R ³ R ⁴ ; and

A = -CH ₂ NHCO- or -CONH-, n = o = p = q = 0 and Y = -H.

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. A summary of all previously known basic structures of archaebacterial lipids is contained in Langworthy and Pond (System Appl. Microbiol. 7, 253-257, 1986). The naturally occurring 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. For this, 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. Since a possible intended use of the lipids according to the invention is in the formation of liposomes or lipid agglomerates for pharmacotherapeutic and / or gene therapeutic applications, 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 are described in detail below.

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.

S ¹ and S ² , which may be the same or different, are represented by the following formula:

-A- (X ¹ ) _r -B- (X ² ) _q -Y

in which mean: A -CONR ¹ -, -COO-, -CH ₂ 0-, -CH ₂ OCO-, -CH ₂ OCOO-, -CH ₂ NR ¹ CO-,

-CH2OCONR ¹ -, -CH ₂ NR ¹ COO-, -CON [(CH ₂ ) _m Y] -, -CH ₂ OCO (CH ₂ ) _m O-, -CONH (CH ₂ ) _m NHCO (CH ₂ ) _m O-, -CH ₂ COO-, -CH ₂ 0 (P0 ₂ R ¹ ) O- or -CH ₂ S-,

B - [(CH ₂ ) _m O] _n -, - [0 (CH ₂ ) _m ] _n -, - [NR ¹ COCHN (R ¹ ) ₂ ] ₀ - or - (NR ⁶ ) _P -,

Y -H, -R ² , -NR ² R ³ , -N ⁺ R ² R ³ R ⁴ , -N [(CH ₂ ) _m Y] ₂ , -NR ² (CH ₂ ) _m Y, -OR ⁵ , -COR ⁷ ,

R ² to R ⁵ ,

m 1 to 5, where m can assume different values within a group, n 0 to 150,

0 0 to 10, p 0 or 1, q 0 to 5 and r 0 or 1,

where in each case 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.

According to the invention, it is preferred that S ¹ and S ² independently of one another represent one of the following groups:

-CH ₂ OP0 ₃ R ¹ R ² , -CH ₂ OP0 ₃ R ¹ - (CH ₂ ) _m -Y, -COOR ³ , -CH ₂ 0-COOR ³ , -CH ₂ OR ³ , -CONH-D, - CH ₂ 0-CONH-D, -CH ₂ 0-CO- (CH ₂ ) _m -0-D, -CONH- (CH ₂ ) _m -NHCO- (CH ₂ ) _m -0-D, -CH ₂ 0 -D, -COO-D, -CON [(CH ₂ ) _m -Y] -D;

in which mean:

D - (CH ₂ CH ₂ 0) _n -E, - (CH ₂ ) _m -Y, - (CH ₂ ) _m -NHCO-CHY- (CH ₂ ) _m -Y,

- (CH ₂ ) _m -N [(CH ₂ ) _m -Y] ₂ , - (CH ₂ ) _m -NR ² - (CH ₂ ) _m -Y;

E -R ⁴ , - (CH ₂ ) _m -NHCO-Z, - (CH ₂ ) _m -NR ⁴ ligand, - (CH ₂ ) _m -CO ligand,

- (CH ₂ ) _m NHCO (CH ₂ ) _m CO ligand, - (CH ₂ ) _m -Y;

Y -NR ⁴ R ^a , -N ⁺ RR ³ R ^b , -NH-C (NH) -NH ₂ ;

- (CH ₂ ) _S - CO ligand,

R ¹ to R ⁶ independently of one another -H, -CH ₃ , -C ₂ H ₅ ;

m 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:

S ¹ = -CONH-D, -COO-D or -CONH- (CH ₂ ) _m -NHCO- (CH ₂ ) _m -0-D and S ² = -COOR ³ ; or

S ¹ = -CH ₂ 0-CONH-D, -CH ₂ 0-D or -CH ₂ 0-CO- (CH ₂ ) _m -0-D and S ² = -CH ₂ OR ³ .

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 .

Preferred examples of the groups represented by R ² to R ⁵ , R ⁷ and R ⁸ include the preferred groups mentioned for R ¹ and the group - (C = N ⁺ H ₂ ) 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.

According to a particularly preferred embodiment of the invention, S ¹ and S ^{2 are} independently selected from the following groups:

- COOH, - COOCH ₃ , - COOC ₂ H _{5 f} - CH ₂ - -OP0 ₃ H ₂ ,

0 - C - N [(CH ₂ ) ₃ - (CH ₃ ) 2] ₂ .

Very particularly preferred combinations of S ¹ and S ² include:

S ¹ = -CH ₂ OCONH (CH ₂ CH2θ) _n CH3 and S ² = S ¹ or -CH ₂ OR ³ ,

S ¹ = -CH ₂ 0 (CH ₂ CH ₂ 0) _n CH ₃ and S ² = S ¹ or -CH ₂ OR ³ ,

S ¹ = -CH ₂ OCO (CH ₂ ) _m O (CH ₂ CH ₂ 0) _n CH ₃ and S ² = S ¹ or -CH ₂ OR ³ or -CH ₂ OCOOR ³

S ¹ = -COO (CH ₂ CH ₂ 0) _n CH ₃ and S ² = S ¹ or -COOR ³ ,

S ¹ = -CONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NH ligand and S ² = S ¹ or -COOR ³ ,

S ¹ = -CONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO (CH ₂ ) _m CO ligand and S ² = S ¹ or -COOR ³ ,

S ¹ = -CONH (CH2) _m NHCO (CH ₂ ) _m O (CH ₂ CH ₂ 0) n (CH ₂ ) mCO ligand and S ² = S ¹ or -COOR ³ ,

S ¹ = -CH ₂ OCONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NH ligand and S ² = S ¹ or -CH ₂ OR ³ ,

S ¹ = -CH ₂ OCONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO (CH ₂ ) _m CO ligand and S ² = S ¹ or -CH ₂ OR ³ ,

S ¹ = -CONH (CH ₂ ) _m NHCO (CH ₂ ) _m O (CH ₂ CH ₂ 0) _n (CH ₂ ) mNHCO-Z and S ² = S ¹ or -COOR ³ ,

S ¹ = -CONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO-Z and S ² = S ¹ or -COOR ³ ,

S ¹ = -CH ₂ OCONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO-Z and S ² = S ¹ or -CH ₂ OR ³ ,

S ¹ = -CONH (CH ₂ ) _m NHCO (CH2) mO (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO-Z and S ² = S ¹ or -COOR ³ , S ¹ = -CONH (CH ₂ CH2θ) _n (CH ₂ ) _m NHCO-Z and S ² = S ¹ or -COOR ³ ,

S ¹ = -CH ₂ OCONH (CH ₂ CH ₂ 0) n (CH2) _m NHCO-Z and S ² = S ¹ or -CH ₂ OR ³ ,

S ¹ = -CONH (CH ₂ ) _m NHCO (CH ₂ ) _m O (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO-Z and S ² = S ¹ or -COOR ³ ,

S ¹ = -CONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO-Z and S ² = S ¹ or -COOR ³ , and

S ¹ = -CH ₂ OCONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO-Z and S ² = S ¹ or -CH ₂ OR ³ .

In each case 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 _k - V - W | -

in which mean:

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

(b) a polyethylene / propylene glycol chain with at least 3 ethylene / - propylene glycol units, and / or

(c) a polyethylene / propylene amine chain with at least 3 ethylene / propylene amine units, and / or

(d) a carbohydrate chain, and / or

(e) an oligopeptide chain and / or

(f) a polylactide chain,

k 0 or 1 and

I 0 or 1.

Preferred linking groups between lipid and ligand are listed below.

where * is a divalent linking group. Preferred examples of 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 oligopeptide chain with 5 to 50 amino acids, preferably with 5 to 15 amino acids, (f) a polylacfide chain, (g) a poly (acrylmorpholine) chain, (h) a poly (vinylpyrrolidone) chain and (i) one poly (acrylamide) chain. Particularly preferred examples of the divalent linking group include the groups -CO (CH ₂ ) ₈ CO-,

- (C = N ⁺ H ₂ ) (CH ₂ ) 2SS (CH ₂ ) 2 (C = N ⁺ H ₂ ) -, -CO (CH ₂ ) ₆ SS (CH ₂ ) ₅ CO-, -COCH (OH) CH (OH) CO-, -CO (CH ₂ ) ₂ COO (CH ₂ ) ₂ OCO (CH ₂ ) ₂ CO- and -COO (CH ₂ ) ₂ S0 ₂ (CH ₂ ) ₂ OCO-.

The materials that can be used to introduce a spacer into the tetraether lipids of the invention include commercially available products. For example, 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. 22337ZZ), DMA (Product No. 20663ZZ), DMP (Product No. . 21666ZZ), DMS (Product No. 20700ZZ), DPDPB (Product No. 21702ZZ), DSG (Product No. 20593ZZ), DSP (Product No. 22585ZZ), DSS (Product No. 21555ZZ), DST (Product No. 20589ZZ), DTBP (Product No. 20665ZZ), DTME (Product No. 22335ZZ), DTSSP (Product No. 21578ZZ), EDC (Product No. 22980ZZ), EGS (Product No. 21565ZZ), EMCA (Product No. 22306ZZ), EMCS (Product No. 22308ZZ), GMBS (Product No. 22309ZZ ), KMUA (Product No. 22211ZZ), LC-SMCC (Product No. 22362ZZ), LC-SPDP (Product No. 21651ZZ), MBS (Product No. 22311ZZ), MSA (Product No. 22605ZZ), PMPI (Product No. 28100ZZ), SATA ( Product No. 26102ZZ), SATP (Product No. 26100ZZ), SBAP (22339ZZ ), SIA (product no. 22349ZZ), SIAB (Product No. 22329ZZ), SMCC (Product No. 22360ZZ), SMPB (Product No. 22416ZZ), SMPH (Product No. 22363ZZ), SMPT (Product No. 21558ZZ), SPDP (Product No. 21857ZZ), Sulfo-BSOCOES (Product No. 21556ZZ), Sulfo-LC-SMPT (Product No. 21568ZZ) and TFCS ( Product No. 22299ZZ) from PIERCE (the product numbers were taken from the product catalog 1999/2000).

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. On the one hand, the opsonization of the vesicles is prevented and thereby the recognition by the reticuloendothelial system. On the other hand, the removal of the individual lipids is suppressed by HDL complexes. The PEG chains preferably have molecular weights of 2000, 3400 or 5000 mass units.

It has been shown that 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.

Thanks to the membrane-spanning macro cycle, 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).

Furthermore, in contrast to the pegylated TEL in serum, an increase in the release compared to the control is observed in liposomes in which conventional pegylated lipids are integrated. Regarding the release properties, pegylated TEL also represent a clear improvement of the conventional PEG system (see 3.3.2, Fig. 8b).

It has been shown that the circulation time of liposomes can be significantly increased by incorporating mono- or bis-pegylated tetraether lipid derivatives.

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. In addition, 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.

By incorporating this lipid, the circulation time of liposomes can be increased significantly. Alternatively, the PEG chains can be replaced by poly (acrylic morpholine), poly (vinyl pyrrolidone) or gangliosides.

With pegylated liposomes carrying cell-specific ligands, the removal of the lipid-ligand conjugates from the liposome membrane is a problem. An improvement can be achieved if the target-specific ligands are coupled to the PEG ends of the pegylated TEL. Because the TEL derivatives are better anchored due to the membrane-spanning lipids. This represents another advantage of pegylated tetraetheriipids when used in target-specific liposomes.

Ligaπd-hydrophilic polymer-Y- (X) _q -B- (X ¹ ) _r A

The tetraether lipid-ligand conjugates according to the invention are outstandingly suitable for cell-specific targeting with the aid of long circulating liposomes.

The 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. Among the carbohydrates, particular preference is given, for example, to galactose, lactose, mannose or sialyl-Lewis-X. According to the invention, 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.

In a preferred embodiment, 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.

In another preferred embodiment, 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. As already mentioned, 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. In the 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 (e.g. tetraethers modified with acid chloride groups or with hydroxymethyl groups) 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.

Methods of making liposomes are well known. In general, the 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. For medical-pharmaceutical purposes, for example, 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.

By shaking by hand, large multilamellar vesicles with a size distribution in the μm 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.

The following methods are considered suitable for the preparation of unilamellar liposomes of a defined size as suitable for the production of liposomes from tetraether lipid derivatives according to the invention:

(a) Ultrasound treatment: The suspension of multilamellar liposomes is 5 at 20 kHz

Minutes (Branson Sonifer B 15). Liposomes with a diameter of around 500 nm are formed.

(b) Extrusion through a French pressure cell: The suspension of multilamellar liposomes is at 16000 p.s.i. extruded four times in the French pressure cell (SLM-Aminco Inc., Urbana IL, USA). Liposomes with a diameter of around 120 nm are formed.

(c) Extrusion through polycarbonate filters: The suspension of multilamellar liposomes is extruded through polycarbonate filters of defined pore size (LiposoFast ^m , Avestin, Ottawa, Canada). When using filters with a pore size of 100 or 200 nm, liposomes are formed which have a size distribution slightly above the pore size. Extrusion through filters of this small pore size can simultaneously serve as sterile filtration, provided all other requirements for sterile filtration (eg exact separation of the non-sterile and sterile sides of the filtration apparatus) are met. A few preparations can be made to facilitate the actual filtration: (ca) The suspension of multilamellar liposomes is between 1 and 5 at 20 kHz

Minutes (s. (A))

(cb) The suspension of multilamellar liposomes is frozen 1-3 times and thawed again.

(cc) The suspension of multilamellar liposomes according to (ca) or (cb) is prefiltered through a polycarbonate filter with a pore size of 600 to 800 nm.

Following the ultrasound treatment or extrusion, 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. For this purpose, a lipid film is first formed as described above. This is suspended in a detergent-containing buffer system (examples of dialysable detergents: octyl-β-D-glucopyranoside or octyl-β-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. When the detergent is removed by dialysis, 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.

Furthermore, an increased detergent stability and an increased serum stability (ie in vivo-like conditions) could be shown (see 4.4.2 and Fig. 17). In the presence of detergents and in the presence of serum, the liposomes containing TEL show a significantly lower release of carboxyflourescein than conventional liposomes (consisting of phosphatidylcholine).

The increase in stability could already be demonstrated with a 10% lipid layer fraction of the TEL derivatives in the liposomes. Since the stability increases continuously with an increase in the proportion of TEL, 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.

For many applications, including the formulation of pharmaceuticals with acid-labile active ingredients and the transfection of eukaryotic cells, pure TEL derivative liposomes are therefore the method of choice.

Using 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. By adding the TEL to the conventional liposomes, 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. For example, 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®. In a further preferred embodiment, 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. For this purpose, 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. B. contains antibodies for which there are corresponding proteins on the cells to be reached with the therapy, 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 same applies to 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.

In a further embodiment according to the invention, 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.

The 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. In the monomolecular layer, 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.

Examples of surfaces to be coated 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).

The following figures and examples illustrate the invention.

Description of the pictures

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.

Examples

1. Fermentation of Sulfolobus acidocaldarius

The fermentation of Sulfolobus acidocaldarius is carried out as follows: To cultivate the preculture, it is inoculated with an aliquot from a glycerol culture that is stored at -80 ° C. This preculture is then shaken at 125 revolutions / minute, 78 ° C., pH = 3.3 for about 50 hours in a shaking bath. With this preculture, the fermenter is now inoculated, which contains sterile medium. An oxygen partial pressure of 20% is set in the fermenter and is kept constant throughout the fermentation. The temperature and the pH value correspond to the values of the preculture and also remain constant during the fermentation. After a fermentation time of 24 hours under these conditions, a continuous addition of nutrients (yeast extract and protein hydrolyzate) begins until the end of the fermentation. This addition ensures a constant optimal supply of the bacteria with nutrients. This leads to faster growth and a high biomass yield. The fermentation is complete after about 50 to 60 hours. After the fermentation has ended, the medium is cooled to room temperature and the biomass is separated from the remaining medium by a separator. This bio-moisturizing substance is then largely freeze-dried by means of freeze-drying. The lyophilisate obtained from this is now the starting point for the subsequent workup steps for isolating the tetraether lipids.

2. Obtaining tetraetheriipids from Sulfolobus acidocaldarius

2.1 Extraction of lipids from Sulfolobus acidocaldarius

53.2 g of lyophilisate are extracted in 3 batches of approx. 18 g each: the freeze-dried cell mass is finely ground, filled into an extraction thimble and covered with glass wool. The extraction takes place in a 150 ml SoxhIett apparatus with 300 ml chloroform / methanol (1: 1 or 2: 1) (500 ml flask). Some of the lipids precipitate out of the brownish-clear extraction solution. The amount of solvent is checked once a day and refilled if necessary (approx. 50 ml every 2 days). After 100-120 h, the solvent is removed on a rotary evaporator and the amount extracted is determined (3.14 g, 5.9%).

2.2 Hydrolysis of the lipids from Sulfolobus acidocaldarius

(a) Variant 1

3.14 g of crude lipid extract are mixed with 350 ml of methanol / 37% hydrochloric acid (4: 1) and then heated under reflux for 48 h. Then 35 ml of 37% hydrochloric acid are added and the mixture is heated for a further 60 h. The precipitate is filtered off. The filtrate is extracted with 150 ml of chloroform and the hydrochloric acid phase twice more with 100 ml of chloroform. The combined organic phases are used to dissolve the filtered precipitate. After the residual water has been separated off, it is dried over sodium sulfate and the solvent is removed. 1.90 g (60%) of hydrolyzate are obtained. (b) Variant 2

Approximately 1.3 g of crude lipid extract are mixed with 300 ml of chloroform / methanol / 37% hydrochloric acid (2: 3: 1; single phase) and then heated under reflux for 48 h. A phase separation is initiated by adding 300 ml of water and the organic phase is separated off. The aqueous phase is extracted twice with 50 ml of chloroform. The combined organic phases are evaporated. 0.52 g (40%) of hydrolyzate are obtained.

The following tetraether lipids are obtained from the Sulfolobus acidocaldarius: Open DGTE: approx. 3% of the total lipid portion

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.

Open DGTE (1)

DGTE (example with four pentacycles) (2)

GCTE (two different head groups):

GCTE (3)

GCTE (4)

analytics:

Thin layer chromatography (silica gel Si 60, Merck):

Chloroform / diethyl ether (9: 1) DGTE R _f = 0.17

GCTE R, = 0.00 Chloroform / methanol (9: 1) DGTE R _f = 0.80

GCTE R, = 0.19

2.3 Separation of DGTE and GCTE by column chromatography

Construction of the column,

80 x 4 cm: 0.5 cm sea sand

61 cm Si 60 (0 0.063-0.200), 400 g (770 ml) 1 cm Celite® 1 cm sea sand glass wool or frit

The silica gel is swollen in chloroform / diethyl ether (9: 1).

3.0 g of hydrolyzate are dissolved in 10 ml of chlorofrom / diethyl ether (9: 1) and applied. 20 ml of eluent are used for rinsing. Then it is chromatographed with chloroform / diethyl ether (9: 1).

Chromatography process:

(Abbr .: chloroform: C, CHCI ₃ ; diethyl ether: E, Et ₂ 0; methanol: M, MeOH)

Conversion of the eluent to CHCI ₃ / MeOH (9: 1)

2.4 Cleaning DGTE

a) Pre-cleaning by gel chromatography:

258 mg of the DGTE fraction from (Section 2.3) are purified on a gel column.

Column: 0 1.8 cm, height 54 cm, 24 g Sephadex LH-20 solvent: chloroform / methanol (1: 1), 2.5 ml / 8 min The fractions of approx. 30 - 45 ml are collected. The UV-active impurities mainly contained in later fractions are separated. 120 mg of pre-purified DGTE are obtained.

b) Final purification of DGTE by silica gel chromatography:

120 mg of pre-purified DGTE (from a)) are chromatographed on a silica gel column.

Column: 0 1.7 cm, height 23 cm, 20 g silica gel Si 60 (0.063-0.200 mm; from Merck) Eluent: cyclohexane / ethyl acetate (4: 1), 3 ml / 6 min

The fractions of approx. 35-65 ml are collected. 68 mg (57%) of clean DGTE are obtained.

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.

3. Pegylated tetraether lipids

3.1 Synthesis of bis (methoxy-PEG) tetraether (II)

DGTE diacid chloride (I) bis (methoxy-PEG) tetraether (II)

9.40 mg (7.10 μmol) DGTE-diacid chloride (I) are mixed with stirring and a nitrogen atmosphere with a solution of 34.1 mg (17.1 μmol) methoxy-PEG-amine (MW = 2000) in 1.1 ml dichloromethane. 50 μl (36.3 mg, 359 μmol) triethylamine are then immediately added and the mixture is stirred for 24 h. Everything that is volatile gets into a cold trap (more fluid Nitrogen) condensed. The residue is chromatographed on silica gel 60 (0.040-0.063 mm; 13 g, mobile phase: chloroform / methanol (8: 2). The fractions containing the product are combined and again on silica gel 60 (0.040-0.063 mm; 37 g, mobile phase: Chloroform / methanol (95: 5) chromatographed.

Yield: 26.6 mg (71%), colorless solid.

analytics:

Thin layer chromatography (Kieselgel 60, Merck):

Flow agent: chloroform / methanol (8: 2); R, = 0.83. Staining: sulfuric acid reagent (brown-black spot) ¹ HN M R spectroscopy:

The integration of the ether protons against the alkyl protons in the ¹ H-NMR spectrum (270 MHz) shows that two PEG chains are bound per tetraether macrocycle: theoretical ratio = 376/152 = 2.47 experimentally determined ratio = 2.56 δ ¹ H (270 MHz, CDCI ₃ ) = 0.7-0.9 (m, alkyl-H), 0.9-1.4 (m, alkyl-H), 1.5-1.8 (m, alkyl-H), 3: 3 -3.9 (m, ether H), 7.01 (t, NH). Selected ¹³ C-NM R spectroscopy data: δ ¹³ C (67.9 MHz, CDCI ₃ ) = 170.7 (-CONH-), 70.6 (-0-CH ₂ -PEG), 72.0, 70.4, 70.0, 69.9, 69.7 (others Ether-C), 59.1 (-CONH-CH ₂ -). Mass spectrometry (MALDI-TOF-MS):

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. The center is approx. M / z = 5335. The limits are / z = 4586 and 6215. The lines can be explained with sodium as adducts of the double-pegylated tetraether (4 pentacycles per tetraether macrocycle). From the center at m / z = 5335 it follows that the PEG chains consist of 44 ethylene oxide units (n = 44) on average. The average molar mass for the compound is therefore MG = 5313.0 g / mol. IR spectroscopy (NaCI): v = 1672, 1524 cnrϊ ¹ (amide l + ll), 1112 (ether). 3.2 Synthesis of methoxy-PEG-tetraether carboxylic acid (III)

DGTE methoxy-PEG-tetraether carboxylic acid (III) n = approx. 46

10.1 mg (7.64 μmol) of diacid are mixed with 1 ml of anhydrous dichloromethane and 1 ml of thionyl chloride under a nitrogen atmosphere. The reaction mixture is heated under reflux for 20 h. The thionyl chloride is then removed under high vacuum and the residue is dried. The brown solid is dissolved in 500 μl of anhydrous dichloromethane under a nitrogen atmosphere and slowly with a solution of 15.3 mg (7.64 μmol) methoxy-PEG-amine (MW = 2000) and 1.70 mg (16.8 μmol) triethylamine, dissolved in 810 μl of anhydrous dichloromethane, added. The mixture is stirred at room temperature for 24 h. Then all volatile constituents are removed under high vacuum and the solid residue is dried. The solid is purified by column chromatography:

1. 5 g SEPHADEX LH-20; Superplasticizer: chloroform

2. 10 g of silica gel (Merck, 0.04-0.063); Mobile phase:

1) chloroform

2) chloroform / methanol (2: 1)

3) chloroform / methanol / water (65: 25: 4)

Yield: 3.0 mg (12%); colorless solid.

analytics:

Thin layer chromatography (Kieselgel 60, Merck):

Eluent: 2-propanol / 35% ammonia solution in water / water (10: 2: 1); R, = 0.43. Staining: sulfuric acid reagent (brown-black spot) ¹ H-NMR spectroscopy:

The integration of the ether protons against the alkyl protons in the ¹ H NMR spectrum (270 MHz) shows that one PEG chain is bound per tetraether macrocycle: δ ¹ H (270 MHz, CDCI ₃ ) = 0.7-0.9 (m, alkyl-H), 0.95-1.45 (m, alkyl-H), 1.45-1.8 (m, alkyl-H), 3.35-3.75 (m, ether-H). Mass spectrometry (MALDI-TOF-MS):

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. The center is around m / z = 3428. The limits are around m / z = 3032 and 3824. The lines can be explained with sodium as adducts of the pegylated tetrahether (approx. 4 pentacycles per tetraether macrocycle). From the center at m / z = 3428 it follows that the PEG chains consist on average of 44 ethylene oxide units (n = 46). The average molar mass for the compound is therefore MG = 3405 g / mol. IR Spectroscopy (KBr): v = 2923cm ^-1 (alkyl), 1733 (carboxyl), 1672 (amide), 1464, 1360 (alkyl), 1113 (ether).

3.3 Biological studies on compound II

3.3.1 Incorporation of Compound II in Liposomes

3.3.1.1 Liposome preparation

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 μl of double-distilled water, incubated for one hour at room temperature, made up to 1 ml by adding 950 μl of double-distilled water and extruded again 20 times through a 100 nm polycarbonate membrane by means of the hand extruder.

3.3.1.2 Verification of incorporation by density gradient centrifugation

Using 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 μl chloroform: methanol (1: 1) (v / v) and placed on silica gel 60 F ₂₅ HPLC plates (Fa. Merck) applied. After developing the HPTLC plates in the eluent chloroform: methanol (2: 1) (v / v), the HPTLC plates are briefly added to the staining solution (3% (w / v) copper acetate in 8% (v / v) phosphoric acid ) immersed and heated at 170 ° C for 30 min. Compound II is identified and quantified by comparison with corresponding lipid standards.

The density gradients of the incorporation experiments show a clear colocalization of compound II with the lipid phosphatidylcholine (Fig. 1). Control experiments show that the free compound, probably as micellar aggregates, can be found at higher densities (insert in Fig. 1). The experiments clearly demonstrate that Compound II can be completely incorporated into liposomes.

3.3.2. Stability studies on liposomes with compound II

3.3.2.1. Preparation of liposomes including carboxyfluorescein

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 μl 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 μl 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). After 5 freeze-thaw cycles (liquid nitrogen - water bath, 37 ° C.), 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).

3.3.2.2. Serum stability of liposomes with compound II

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. In the presence of serum, 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.

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.

4. Phosphorylated tetraether lipids

4.1 Synthesis of DGTE-P (IV)

DGTE DGTE-P (IV)

102 mg (78.7 μmol) DGTE are dissolved in 700 μl anhydrous tetrahydrofuran and 36.3 μl (450 μmol) anhydrous pyridine are added under protective gas (nitrogen or argon). 16.5 μl (180 μmol) phosphoryl chloride in 200 μl tetrahydrofuran are added dropwise (within 5 min) at 0 ° C. using a syringe. The mixture is stirred for 3 h at 0 ° C., mixed with 800 μl of a 10% sodium hydrogen carbonate solution and stirred for a further 15 min. The mixture is brought to pH = 0 with 10% hydrochloric acid while cooling with ice. 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)). The product is dissolved in Chloroform / methanol (2: 1) and 14% hydrochloric acid were taken up and extracted five times with chloroform / methanol (2: 1), the combined organic phases were dried over sodium sulfate, the solvent was removed in vacuo and the solid, slightly yellowish residue became recrystallized from chloroform and chloroform / methanol (2: 1).

Yield: 23 mg (20%), colorless solid.

analytics:

Thin layer chromatography (Kieselgel 60, Merck):

Superplasticizer: chloroform / methanol / water / 37% ammonia solution in water

(60: 34: 5.5: 0.5); R _f = 0.07.

Staining: sulfuric acid reagent (brown-black spot) Mass spectrometry (ESI): m / z = 1452.2 (53) [M ⁺ -1], 725.8 (100) [M ²⁺ -1] IR spectroscopy (NaCI): v = 2923 cm ^"1 (alkyl ), 2855 (alkyl), 1462 (alkyl), 1377 (alkyl), 1115 (ether), 1061 (POC)

4.2 Synthesis of DGTE-PE (V)

DGTE DGTE-PE (V)

150 μl of anhydrous tetrahydrofuran is placed under a nitrogen atmosphere. At 0 to 5 ° C (ice bath) 6.20 μl (68.2 μmol) phosphoryl chloride and then 10.4 μl (74.9 μmol) triethylamine are slowly added via a syringe. With good stirring, 44.3 mg (34.0 μmol) of anhydrous DGTE are slowly added dropwise at 0 ° C. within 15 min. After stirring for 1 h, the reaction mixture is mixed with a solution of 4.50 μl (74.9 μmol) anhydrous ethanolamine, 37.7 μl (270 μmol) triethylamine and 88.6 μl anhydrous tetrahydrofuran. The mixture is stirred at 0-5 ° C for 1 h and then acidified with 1M hydrochloric acid (pH = 2), whereupon a colorless precipitate is formed. 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.

analytics:

Thin layer chromatography (Kieselgel 60, Merck):

Superplasticizer: chloroform / methanol / water / 37% ammonia solution in water

(60: 34: 5.5: 0.5); R, (DGTE-PE) = 0.23.

Flow agent: chloroform / methanol (3: 1); R _f (DGTE) = 0.95, R _f (DGTE-PE) = 0.0.

Staining: sulfuric acid reagent (brown-black spot)

Mass spectrometry (ESI): m / z: 1540.4 (64) [M ⁺ -1], 770.8 (100) [M ²⁺ -1].

IR spectroscopy (NaCI): v = 3139 cm ^"1 (ammonium), 3047 (ammonium), 2921 (alkyl), 2852 (alkyl), 1446 (alkyl),

1406 (alkyl), 1378 (alkyl), 1215 (P = 0), 1074 (P-O-C).

4.3 Synthesis of DGTE-PC (VI) (2 stages)

DGTE DGTE-PBr

Tπmethylamin

DGTE-PC (VI)

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

363 mg (1.50 mmol) of bromoethyl dichlorophosphate are dissolved in 4.0 ml of anhydrous dichloromethane under a nitrogen atmosphere and slowly mixed with 306 mg (3.03 mmol) of triethylamine. 98.4 mg (75.6 μmol) of DGTE, dissolved in 4.0 ml of anhydrous dichloromethane, are added dropwise to the reaction mixture with stirring in the course of 2 h. The mixture is stirred for 3 days with the exclusion of light and under a nitrogen atmosphere at room temperature. 25 ml of a chloroform / methanol mixture (2: 1) and 10 ml of semi-saturated sodium chloride solution are added to the reaction mixture. 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:

1. 20 g SEPHADEX LH-20; Superplasticizer: chloroform / methanol (2: 1)

2. 5 g silica gel (0.04-0.063); Superplasticizer: chloroform / methanol (2: 1)

Yield: 104 mg (82%), colorless solid.

analytics:

Thin layer chromatography (Kieselgel 60, Merck):

Eluent: eluent: chloroform / methanol (2: 1); R / (DGTE-PBr) = 0.19 mass spectrometry (ESI -): m / z = 1666.2 (M-1), 1584.4 (-HBr), 832.8 (M-2).

2nd stage: synthesis of DGTE-PC (VI)

104 mg (62.3 μmol) 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 combined organic phases are dried over sodium sulfate. The solvent is removed in vacuo and the brown oil is dried in a high vacuum. The purification is carried out via column chromatography:

1. 20 g SEPHADEX LH-20; Mobile solvent: chloroform / methanol (2: 1)

2. 5 g silica gel (0.04-0.063); Superplasticizer: chloroform / methanol / 35% ammonia solution in water / water (60: 35: 5.5: 0.5).

Yield: 29.1 mg (29%), colorless solid.

analytics:

Thin layer chromatography (Kieselgel 60, Merck):

Superplasticizer: chloroform / methanol / 35% ammonia solution in water / water

(60: 35: 5.5: 0.5); R, (DGTE-PC) = 0.02; R, (DGTE-PBr) = 0.71

Mass Spectroscopy (ESI +):

/ ?? / z = 1624.55 (M + 1), 813.03 (M + 2).

IR Spectroscopy (NaCI): v = 2923, 2855 cm ^-1 (alkyl), 1462, 1378 (alkyl), 1236 (P = 0), 1089 (POC).

4.4 Biological studies on compounds V and VI

4.4.1. Production and characterization of liposomes from V

4.4.1.1. Liposome preparation by detergent dialysis

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 μl dialysis buffer (10 mM HEPES, pH 7.4, 150 mM NaCI), transferred into a dialysis capsule (Carl Roth GmbH, Karlsruhe, Germany) and provided with a dialysis membrane (exclusion limit 10 kDa, Dianorm GmbH, Munich). 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.

4.4.1.2. Characterization of the liposomes from V

The composition of the liposomes is examined using thin layer chromatography. For the extraction, 20 μl of the liposome solution are mixed with 80 μl 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 μl chloroform: methanol (2: 1) and applied to an HPTLC thin-layer plate (Merck, Darmstadt, Germany). 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 μl. The particle size is weighted based on a Gaussian distribution.

After the in 1.1. described processes, 4 liposome batches were prepared with the following contents of compound V: 20 mol%, 33 mol%, 50 mol%, 100 mol%. Since the compound V completely spans the membrane, but phosphatidylcholine only half, the half sides of the lipid bilayer have the following layer composition: 33%, 50%, 66% and 100% compound V.

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).

4.4.2. Stability studies on liposomes from V and VI

4.4.2.1. Preparation of liposomes including carboxyfluorescein

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).

After 5 freeze-thaw cycles (liquid nitrogen - water bath, 37 ° C.), 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.

The analysis of the lipid composition is carried out analogously to application example 4.4.1.2. The following solvents are used in the HPTLC:

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.

4.4.2.2. Stability studies in biological environments

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.

To measure the detergent stability, 25 μl of the liposome sample is mixed with 75 μl 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).

To measure serum stability, 60 μl liposomes are mixed with 240 μl fetal calf serum and incubated at 37 ° C. for 16 h. The fluorescence intensity is measured every hour using the parameters described above. 60 μl of a liposome solution, which is mixed with 240 μl 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. Both in the presence of detergents and in the presence of serum, 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.

4.4.3. Liposomes from compound VI as an oral drug delivery system

4.4.3.1. Incorporation of octreotide in compound VI liposomes

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 μmol of compound VI and 20 μmol 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.). After determining the lipid content by phosphate determination, 15 μmol lipid are transferred to a reaction vessel, frozen at -70 ° C. and lyophilized. For incorporation, 2 mg octreotide are dissolved in 20 μl PBS and the lyophilisate is hydrated with this solution. After complete hydration, the liposome suspension is diluted to 1 ml. To separate the non-incorporated ocreotide, 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 μl 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:

320 ml disodium hydrogen phosphate solution (50.4 mM)

76 ml sodium dihydrogen phosphate solution (23.0 mM)

600 ml HPLC water with 10% phosphoric acid to pH = 7.4 (pH meter) bring column: GROM SiL 100 ODS-0 AB, 5 μm, 250 * 4.6 mm

Guard column: GROM-SiL 120 Butyl-1 ST, 5 μm, 20 * 4.6 mm flow: 1 ml / min

Detection: UV 210 nm Temperature: room temperature

4.4.3.2. Oral ingestion of octreotide after formulation with compound VI

Oral uptake of octreotide, previously incorporated into PC and Compound VI liposomes, is tested in a rat model and compared to oral uptake of free octreotide.

In each case, 100 μg octreotide (as a liposomal formulation or free) is administered orally into the stomach using a gavage. At various times (30, 60, 120, 300, 420 min), 200 μl 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.

5. Cationic tetraether lipids

5.1 Synthesis of AF4 (IX) (3 stages) 1st stage: synthesis of d / -BOC-TREN (VII)

1.40 ml (9.35 mmol) of TREN [tris (2-aminoethyl) amine] and 3.91 ml (28.1 mmol) of triethylamine are dissolved in 40 ml of anhydrous tetrahydrofuran under a nitrogen atmosphere. 4.61 g (18.7 mmol) of BOC-ON are also dissolved in 10 ml of anhydrous tetrahydrofuran and sprayed into the solution via a septum and with water cooling. The reaction mixture is stirred at room temperature for 19 h. 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.

Yield: 561 mg (17%), yellow oil.

analytics:

Thin layer chromatography (Kieselgel 60, Merck):

Plasticizer: ethyl acetate; R _f = 0.12.

Staining: ninhydrin solution (red spot)

Mass spectrometry (FD ⁺ ): m / z: 347.7 [M ⁺ +1]

¹ H-NM R spectroscopy (270 MHz, CDCI ₃ ): δ = 1.43 (s, 18 H. 2 x .Bu), 2.45-3.25 (m, 12 H, CH ₂ ) Stage 2: Synthesis of BOC-AF4 (VIII) (BOC-protected IX)

DGTE diacid chloride BOC-AF4 (VIII)

11.1 mg (8.17 μmol) of DGTE diacid chloride are mixed with 1.0 ml of anhydrous dichloromethane under a nitrogen atmosphere. As soon as the acid chloride is dissolved, 11 μl (78 μmol) triethylamine and 29.7 mg (85.7 μmol) oY-BOC-TREN, dissolved in 0.3 ml anhydrous dichloromethane, are added in succession with stirring and ice cooling. After 1 h the cooling is removed and the mixture is stirred for a further 18 h at room temperature. The solvent is evaporated and the residue is chromatographed on silica gel using ethyl acetate (about 5 g of silica gel 60, 0.040-0.063 mm).

Yield: 6.0 mg (37%), colorless oil.

analytics:

Thin layer chromatography (Kieselgel 60, Merck):

Plasticizer: ethyl acetate; R _f = 0.27.

Staining: sulfuric acid reagent (brown-black spot)

Mass spectrometry (FD ⁺ ): m / z: 1979 [M ⁺ ] Synthesis of AF4 (IX)

(a) Variant 1

BOC-AF4 AF4

The BOC-AF7 is dissolved in chloroform and stirred for 15 minutes with trifluoroacetic acid (TFA) to remove the BOC protective groups.

(b) Variant 2

BOC-AF4 AF4 (IX)

5.00 mg (2.50 μmol) BOC-AF4 are dissolved in 1 ml anhydrous dichloromethane under a nitrogen atmosphere. 240 μl (3.30 mmol) of acetyl chloride are carefully sprayed into 1 ml of anhydrous methanol (dried over Mg) at 0 ° C. and under a nitrogen atmosphere. After stirring for 20 min, the methanolic hydrogen chloride solution is added to the BOC-AF4 solution at 0 ° C. After stirring for a further 30 min at room temperature, the solvent is removed in vacuo. A colorless salt remains which is completely soluble in chloroform / methanol 1: 1. The substance is taken up twice more in chloroform / methanol and concentrated again.

Yield: 3.0 mg (%), colorless oil. analytics:

Thin layer chromatography (Kieselgel 60, Merck):

Mass spectrometry (ESI): m / z = 1578.6 ([M + 1] ⁺ ), 790.0 ([M + 2] ²⁺ ), 527.0 ([M + 3] ³⁺ ), 395.4 ([M + 4] ⁴⁺ ).

5.2 Synthesis of AF6 (X)

DGTE diacid chloride AF6 (X)

21 mg (16 μmol) of DGTE diacid chloride are mixed with 2.0 ml of anhydrous dichloromethane under a nitrogen atmosphere. As soon as the acid chloride is dissolved, 180 μl (800 μmol) bis- [3- (dimethylamino) propyl] amine are added with stirring and ice cooling. After 30 min, the cooling is removed and the mixture is stirred at room temperature for a further 18 h. The light yellow solution is mixed with 10 ml of chloroform / methanol (4: 1) and washed with 10 ml of water. Then the solvent is evaporated and the residue is chromatographed on silica gel (about 5 g of silica gel 60, 0.040-0.063 mm). Chloroform / methanol / glacial acetic acid / water (9: 4: 1: 1) is used as the eluent.

Yield: 16 mg (60%), colorless oil.

analytics:

Thin layer chromatography (silica gel 60):

Flow agent: chlorine form / methanol / acetic acid / water (9: 4: 1: 1); R _f = 0.1. Staining: sulfuric acid reagent (brown-black spot) Mass spectrometry (DEI):

M ⁺ = 1661 IR spectroscopy (NaCl): v = 1649 cm ^'1 (amide I), 1461 and 1377 (alkyl).

5.3 Synthesis of AF7 (XII) (2 stages)

1st stage: synthesis of BOC-AF7 (XI) (BOC-protected XII)

tr / BOC-spermine

DGTE diacid chloride BOC-AF7 (XI)

10.6 mg (7.79 μmol) of DGTE diacid chloride are mixed with 1.0 ml of dichloromethane under a nitrogen atmosphere. As soon as the acid chloride is dissolved, 11 μl (78 μmol) triethylamine and 39.1 mg (77.8 μmol) tri-OC spermine, dissolved in 0.2 ml CH ₂ Cl ₂ , are added in succession with stirring and ice cooling. After 30 min, the cooling is removed and the mixture is stirred at room temperature for a further 18 h. The solvent is removed in vacuo and the residue is dissolved in t7-hexane / ethyl acetate (1: 1). 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). In the first chromatography, t? -Hexane / ethyl acetate (1: 1) is used as the eluent, the second is carried out with pure ethyl acetate.

Yield: 4.2 mg (24%), colorless oil.

analytics:

Thin layer chromatography (silica gel 60):

Eluent: n-hexane / ethyl acetate = 3: 4; R ^ = 0.2. Essigester; R _f = 0.7. Staining: sulfuric acid reagent (brown-black spot) mass spectrometry (FD ⁺ ): m / z: 2291, 5 [M ⁺ ], 501.7 [(.77-BOC-Spermin ⁺ -1]

2nd stage: synthesis of AF7 (XII)

BOC-AF7 (XI) AF7 (XII)

1.9 mg (0.83 μmol) 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.

5.4. Biological studies on the compounds IX and X

5.4.1. Production of liposomes from the compounds IX and X

The liposomes are prepared by detergent dialysis according to the method described in 4.4.1.1. described method. In addition to the compound IX or X, 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.

5.4.2. Transfection of animal cells with liposomes from X

Chinese hamster ovary cells (CHO cells) are sown 24 h before transfection with a density of 3x10 ⁵ x cm ^"2 in 48-well cell culture plates. The cultivation takes place in Iscoves Modified Dulbeccos medium with 10% fetal calf serum, 2 mM glutamine . 100 U / ml penicillin and 100 μg / ml streptomycin (37 ° C, 5% C0 ₂ , 70% humidity). 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 μl with 10 mM HEPES, pH 7.4, 150 mM sodium chloride. The batches are mixed with 70 μl Opti-MEM (Life Technologies, Karlsruhe, Germany) and incubated for 30 min at room temperature. 0.15 μg plasmid DNA are added to 70 μl Opti-MEM for each batch and incubated for 15 min at room temperature. The CHO cells are washed twice with PBS, 150 μl 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 μl of culture medium and incubated for a further 24 h.

Two different plasmids are used which code for the reporter genes β-galactosidase or the green fluorescent protein (GFP) (pCMVß or pEGFP-N2; Clontech Inc., Palo Alto, CA, USA). To determine the transfection efficiency, the β-galactosidase activity expressed in the cells is determined. For this purpose, the cells in the 48-well plates are washed three times with PBS, with 150 μl bidist. Water was added, frozen at -70 ° C. and thawed again and then centrifuged at 3000 g for 30 min. 10 μl of the β-galactosidase determination are fed in from the cell lysate in the supernatant (Groth et al., Anal. Biochem. 1998, 258, 141-143.). The Zeil protein is determined using the bichinolin method on 20 μl of the cell lysate with serum albumin as standard (Smith et al., Anal. Biochem. 1985, 150, 76-85.). The specific ß-galactosidase activity is given in mU / mg cell protein.

Transfections with the green fluorescent protein are examined by fluorescence microscopy (Zeiss Axiovert 25 microscope with fluorescence excitation: filter LP 515, beam splitter FT 510, Carl Zeiss, Jena, Germany; digital microscope camera: DMC 1e, Polaroid Corporation, Cambridge, MA, USA )

Compound X can be used to produce the three co-lipids phosphatidylcholine (PC), cholesterol (Chol) and dioleylphosphatidylethanolamine (PE) in different ratios. 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. As an example, 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 / μg DNA. In the presence of 5% fetal calf serum in a transfection approach, the maximum transfection efficiency drops by approx. 30% and the optimal lipid-DNA ratio shifts to 7.5 nmol X / μg DNA. The transfection experiments with the green fluorescent protein show that approx. 10% of the CHO cells are transfected (Fig. 5). The serum stability is remarkable and represents a significant improvement over the compounds AF1 and AF + based on tetraetheriipids (cf. 5.4.3.). The compounds AF1 and AF + correspond to the compounds (B) and (C) described in WO-A-99/10337.

5.4.3. Transfection of animal cells with liposomes from IX

The experiments with liposomes containing compound IX are carried out analogously to 5.4.2. carried out. Liposomes from IX basically show the same transfection behavior as liposomes from X. The transfection optimum is around 7.5 nmol / μg DNA. Compared to the existing transfection agents based on tetraetheriipids (AF + and AF1), compound IX represents a significant improvement. As shown in Fig. 6, compound IX is 4-5 times more efficient than AF1. In addition, 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.

Claims

claims

1. Tetraether lipid derivative represented by one of the following general formulas (1) to (6):

-A- (X ¹ ) _r -B- (X ² ) _q -Y

in which mean:

-CONR ¹ -, -COO-, -CH ₂ 0-, -CHzOCO-, -CH ₂ OCOO-, -CH ₂ NR ¹ CO-, -CH ₂ OCONR ¹ -, -CH ₂ NR ¹ COO-, -CON [(CH ₂ ) _m Y] -, -CH ₂ OCO (CH ₂ ) _m O-, -CONH (CH ₂ ) _m NHCO (CH ₂ ) _m O-, -CH ₂ COO-, -CH ₂ 0 (P0 ₂ R ¹ ) 0- or -CH ₂ S-, 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 -,

Y -H, -R ² , -NR ² R ³ , -N ⁺ RR ³ R ⁴ , -N [(CH ₂ ) mY] ₂ , -NR ² (CH ₂ ) _m Y, -OR ⁵ ,

-COR ⁷ , -NHC (NH) NH ₂ , -NHCOY or -SR ⁸ , where Y can have different meanings within a group,

R ² to R ⁵ ,

m 1 to 5, where m can assume different values within a group, n 0 to 150,

0 0 to 10,

P 0 or 1, q 0 to 5 and r 0 or 1,

and salts of these compounds,

A = -CH ₂ NHCO- or -CONH-, n = o = p = q = 0 and Y = -H.

The tetraether lipid derivative according to claim 1, wherein

S ¹ and S ² independently of one another represent one of the following groups: -CH ₂ OP0 ₃ R ¹ R ² , -CH ₂ OP0 ₃ R ¹ - (CH ₂ ) _m -Y, -COOR ³ , -CH ₂ 0-COOR ³ , -CH ₂ OR ³ , -CONH-D, -CH ₂ 0-CONH-D, -CH ₂ 0-CO- (CH ₂ ) _m -0-D, -CONH- (CH ₂ ) m-NHCO- (CH ₂ ) _m -0-D, -CH ₂ 0-D, -COO-D, -CON [(CH ₂ ) _m -Y] -D;

in which mean:

- (CH ₂ ) _m -N [(CH ₂ ) _m -η ₂ , - (CH ₂ ) _m -NR ² - (CH ₂ ) _m -Y;

- (CH ₂ ) _m NHCO (CH ₂ ) _m CO ligand, - (CH ₂ ) _m -Y; Y -NR R ⁵ , -N ⁺ R ⁴ R ⁵ R ⁶ , -NH-C (NH) -NH 2,

- (CH ₂ ) _S - CO ligand,

R ¹ to R ⁶ independently of one another -H, -CH ₃ , -C ₂ H ₅ ;

m is an integer from 1 to 4 and s is an integer from 1 to 4.

3. tetraether lipid derivative according to claim 2, wherein S ¹ -CONH-D, -COO-D, -CONH- (CH ₂ ) _m -NHCO- (CH ₂ ) _m -0-D and S ^{2 is} -COOR ³ .

4. tetraether lipid derivative according to claim 2, wherein S ^{1 is} -CH ₂ 0-CONH-D, -CH ₂ 0-D, -CH ₂ 0-CO- (CH ₂ ) _m -0-D and S ^{2 is} for -CH ₂ OR ³ stands.

5. tetraether lipid derivative according to any one of claims 2 to 4, wherein D - (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 , - (CH ₂ ) ₄ -NR ² - (CH ₂ ) ₃ -Y.

6. tetraether lipid derivative according to any one of claims 2 to 5, wherein D - (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 ] - (CH ₂ ) ₃ -N (R ⁵ ) ₂ , - (CH ₂₎ 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 , - (CH ₂ ) ₄ -NH- (CH ₂ ) ₃ -N ⁺ (R ⁵ ) ₃ .

7. tetraether lipid derivative according to any one of claims 2 to 5, wherein E - (CH ₂ ) ₂ -Y, -CH ₃ , - (CH ₂ ) ₂ -NHCO-Z, - (CH ₂ ) ₂ -NR ⁴ ligand or - (CH ₂ ) _{m is} -CO ligand.

8. tetraether lipid derivative according to one of claims 1 to 5 or 7, wherein Y is one of the groups -NHR ⁵ , -N (R ⁵ ) ₂ , -N ⁺ (R ⁵ ) ₃ , -NH-C (NH) -NH ₂ ,

9. tetraether lipid derivative according to claim 8, wherein Y is -NH ₂ , -N (CH ₃ ) ₂ , -N ⁺ (CH ₃ ) ₃ .

10. tetraether lipid derivative according to any one of claims 2 to 9, wherein Z represents one of the following groups:

- (CH ₂ ) ₂ - CO ligand,

11. tetraether lipid derivative according to any one of claims 1 to 10, wherein R ¹ to R ⁶ independently of one another are -H or -CH ₃ .

12. tetraether lipid derivative according to any one of claims 1 to 11, wherein n is a number in the range of 3 to 130.

13. tetraether lipid derivative according to any one of claims 1 to 12, wherein n is a number in the range of 40 to 85.

14. The tetraether lipid derivative according to claim 1, wherein S ¹ and S ^{2 are} independently selected from the following groups:

- COOH, - COOCH ₃ , - COOC2H5, - CH ₂ - OPO3H2,

0 O

-CH ₂ - 0— P— 0— (CH ₂ ) ₂ - N ⁺ H _{3 (} - CH ₂ - 0— P— 0— (CH ₂ ) ₂ - ⁺ (CH ₃ ) ₃

I. 'I. )

0 O

O - CN [(CH ₂ ) ₃ - N (CH ₃ ) 2] ₂ _

15. The tetraether lipid derivative according to claim 1, wherein the spacer is a divalent linking group represented by the following general formula: - U _k - V - W | -

in which mean:

U is a group which is coupled to the lipid and which is selected from the group consisting of -O-, -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 -0-, -S-, -CO-, -OCO-, -NHCO-, -NHCS-, - (CN ⁺ H ₂ ) -, -NH- and o

- N

V (a) a branched or unbranched alkylene chain with 1 to 30

Carbon atoms, the carbon atoms being partially replaced by the

, Groups -S-S-, -CO-, -CH (OH) -, -NHCO-, -CONH-, cyclohexylene,

Phenylene, -COO-, -OCO-, -S0 ₂ -, -0- or -CH (CH ₃ ) - can be replaced, and / or

(d) a carbohydrate chain, and / or

(e) an oligopeptide chain and / or

(f) a polylactide chain, k 0 or 1 and

I 0 or 1.

16. tetraether lipid derivative according to any one of claims 1 to 15, wherein the ligand is selected from the group consisting of proteins, peptides, vitamins, carbohydrates and peptidomimetics.

17. The tetraether lipid derivative according to claim 16, wherein the protein is an antibody, a fragment of an antibody, a lectin or an apo-lipoprotein or a protein binding to a cell surface receptor.

18. The tetraether lipid derivative according to claim 16, wherein the ligand contains 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 peptide sequences from transferrin.

19. The tetraether lipid derivative according to claim 16, wherein the ligand is a vitamin selected from folic acid and vitamin B ₁₂ .

20. tetraether lipid derivative according to claim 1 or 2, wherein S ¹ and S ^{2 are the} same.

21. tetraether lipid derivative according to claim 1 or 2, wherein S ¹ and S ^{2 are} different.

22. The tetraether lipid derivative according to claim 21, wherein S ¹ comprises a cationic group and S ² comprises a ligand.

23. The tetraether lipid derivative according to claim 21, wherein S ¹ comprises a neutral group and S ² comprises a ligand.

24. The tetraether lipid derivative according to claim 21, wherein S ¹ comprises an anionic group and S ² comprises a ligand.

25. The tetraether lipid derivative according to claim 2, wherein S ^{1 is} -CH ₂ OCONH (CH ₂ CH ₂ 0) _n CH ₃ and S ² is S ¹ or -CH ₂ OR ³ .

26. The tetraether lipid derivative according to claim 2, wherein S ^{1 is} -CH ₂ 0 (CH ₂ CH ₂ 0) _n CH ₃ and S ² is S ¹ or -CH ₂ OR ³ .

27. The tetraether lipid derivative according to claim 2, wherein S ^{1 is} -CH ₂ OCO (CH ₂ ) _m O (CH ₂ CH ₂ 0) _n CH ₃ and S ² is S ¹ or -CH ₂ OR ³ or -CH ₂ OCOOR ³ .

28. The tetraether lipid derivative according to claim 2, wherein S ^{1 is} -COO (CH ₂ CH ₂ 0) _n CH ₃ and S ² is S ¹ or -COOR ³ .

29. The tetraether lipid derivative according to claim 2, wherein S ¹ -CONH (CH ₂ CH ₂ 0) _{n is} (CH ₂ ) _m NH ligand and S ² is S ¹ or -COOR ³ .

30. The tetraether lipid derivative according to claim 2, wherein S ¹ -CONH (CH ₂ CH ₂ 0) _{n is} (CH ₂ ) _m NHCO (CH ₂ ) _m CO ligand and S ² is S ¹ or -COOR ³

31. The tetraether lipid derivative according to claim 2, wherein S ¹ -CONH (CH ₂ ) _m NHCO (CH ₂ ) _m O (CH ₂ CH ₂ 0) _{n is} (CH ₂ ) _m CO ligand and S ² means S ¹ or -COOR ^3rd

32. Tetraether lipid derivative according to claim 2, wherein S ^{1 is} -CH ₂ OCONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NH ligand and S ² is S or -CH ₂ OR ³ .

33. Tetraether lipid derivative according to claim 2, wherein S ^{1 is} -CH ₂ OCONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO (CH ₂ ) _m CO ligand and S ² is S ¹ or -CH ₂ OR ³ .

34. Tetraetherlipidderivat according to claim 2, wherein S ¹ -CONH (CH ₂ ) _m NHCO (CH ₂ ) _m O (CH ₂ CH ₂ 0) _{n is} (CH ₂ ) _m NHCO-Z and S ² is S or -COOR ³ ,

35. Tetraetherlipidderivat according to claim 2, wherein S ¹ -CONH (CH ₂ CH ₂ 0) _{n is} (CH ₂ ) _m NHCO-Z and S ² is S ¹ or -COOR ³ .

36. Tetraether lipid derivative according to claim 2, wherein S ^{1 is} -CH ₂ OCONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO-Z and S ² is S ¹ or -CH ₂ OR ³ .

37. Tetraetherlipidderivat according to claim 2, wherein S ¹ -CONH (CH ₂ ) _m NHCO (CH ₂ ) _m O (CH ₂ CH ₂ 0) _π (CH ₂ ) _{m is} NHCO-Z and S ² means S ¹ or -COOR ^3rd

38. Tetraether lipid derivative according to claim 2, wherein S ¹ -CONH (CH ₂ CH ₂ 0) _{n is} (CH ₂ ) _m NHCO-Z and S ² is S ¹ or -COOR ³ .

39. Tetraether lipid derivative according to claim 2, wherein S ^{1 is} -CH ₂ OCONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO-Z and S ² is S ¹ or -CH ₂ OR ³ .

40. Tetraetherlipidderivat according to claim 2, wherein S ¹ -CONH (CH ₂ ) _m NHCO (CH ₂ ) _m O (CH ₂ CH ₂ 0) _{n is} (CH ₂ ) _m NHCO-Z and S ² means S ¹ or -COOR ^3rd

41. Tetraether lipid derivative according to claim 2, wherein S ¹ -CONH (CH ₂ CH ₂ 0) _{n is} (CH ₂ ) _m NHCO-Z and S ² means S ¹ or -COOR ³ .

42. Tetraether lipid derivative according to claim 2, wherein S ^{1 is} -CH ₂ OCONH (CH ₂ CH ₂ 0) _n (CH ₂ ) _m NHCO-Z and S ² is S ¹ or -CH ₂ OR ³ .

43. A composition containing two or more of the tetraether lipid derivatives according to one of claims 1 to 42.

44. Liposome containing one or more tetraether lipid derivatives according to one of claims 1 to 42.

45. Liposome according to claim 44, containing further double-layer-forming cationic, neutral or anionic lipids.

46. Liposome according to claim 45, containing the cationic lipid DOTAP® and / or DOSPER® and / or DC-Chol®.

47. Liposome according to one of claims 44 to 46, containing the neutral lipids phosphatidylcholine and / or phosphatidylethanolamine and / or phosphatidylglycerol and / or phosphatidylserine and / or phosphatidic acid and / or cholesterol and / or sphingomyelin.

48. Lipid agglomerate containing one or more layers of one or more tetraether lipid derivatives according to one of claims 1 to 42.

49. Lipid agglomerate according to claim 48, containing further double-layer-forming cationic, neutral or anionic lipids.

50. Lipid agglomerate according to claim 49, containing the cationic lipid DOTAP® and / or DOSPER® and / or DC-Chol®.

51. Lipid agglomerate according to one of claims 48 to 50, containing the neutral lipids phosphatidylcholine and / or phosphatidylethanolamine and / or phosphatidylglycerol and / or phosphatidylserine and / or phosphatidic acid and / or cholesterol and / or sphingomyelin.

52. Liposome according to one of claims 44 to 47 or lipid agglomerate according to one of claims 48 to 51, wherein the weight ratio of tetraether lipid derivative to further lipids is 5: 1 to 1: 100.

53. Liposome according to one of claims 44 to 47 or 52 or lipid agglomerate according to one of claims 48 to 52, further comprising nucleic acid molecules and / or compounds analogous to nucleic acid molecules and optionally polycations.

54. Liposome or lipid agglomerate according to claim 53, wherein the polycations are selected from polyethyleneimine, poly-lysine and protamine.

55. Pharmaceutical composition containing liposomes according to one of claims 44 to 47 or 52 to 54 or lipid agglomerates according to one of claims 48 to 54 and a physiologically compatible diluent.

56. In vitro transfection kit containing liposomes according to one of claims 44 to 47 or 52 to 54 and / or lipid agglomerates according to one of claims 48 to 54 and suitable buffers.

57. Use of liposomes according to one of claims 44 to 47 or 52 to 54 or of lipid agglomerates according to one of claims 48 to 54 for the manufacture of a medicament for gene therapy in mammals.

58. Use of liposomes according to one of claims 44 to 47 or 52 to 54 or a lipid agglomerate according to one of claims 48 to 54 for the transfection of eukaryotic cells in vitro.

59. Use of liposomes according to one of claims 44 to 47 or 52 to 54 or a lipid agglomerate according to one of claims 48 to 54 for packaging active substances for oral, enteral, parenteral, intravenous, intramuscular, interarticular, topical, subcutaneous, pulmonary or intraperitoneal application.

0. Use of the tetraether lipids according to one of claims 1 to 42 or the composition according to claim 43 for coating surfaces, in particular metal or plastic surfaces.