Novel facultative cationic sterols

Field of the invention

The present invention relates to novel facultative cationic sterols and liposomes comprising such sterols. A particular aspect of the invention relates to amphoteric liposomes comprising the novel facultative cationic sterols.

Background of the invention

Sterol derivatives of the general formula 1 are known in the art and are disclosed in WO 02/066490 by Panzner et al., the content of which is incorporated herein by reference.

Cation - Spacer 2 - Y - Spacer 1 - X - Steryl (1),

wherein the cation is derived from piperazines, imidazoles, morpholines, purines, pyrimidines and /or pyridines;

the spacers 1 and 2 are selected from the group consisting of lower alkyl residues with up to 8 C atoms with a linear, branched or cyclic structure and 0, 1 or 2 ethylenically unsaturated bonds;

the linking groups X and/or Y are selected from the group consisting of -(C=O)-O-, -(C=O)-NH-, -(C=O)-S-, -O-, -NH-, -S-, -CH=N-, -0-(O=C)-, -S-(O=C)-, -NH-(O=C)-, and/or -N=CH-;

the steryl is selected from the group consisting of cholesteryl, sitosteryl, campesteryl, desmosteryl, fucosteryl, 22-ketosteryl, 20-hydroxysteryl, stigmasteryl, 22-hydroxycholesteryl, 25-hydroxycholesteryl, lanosteryl, 7-dehydrocholesteryl, dihydrocholesteryl, 19-hydroxycholesteryl, 5α-cholest-7-en-3β-yl,

7-hydroxycholesteryl, epicholesteryl, ergosteryl, and/or dehydroergosteryl;

and the pka value of the compounds is between 3.5 and 8.

US 5,965,4343 of Wolff et al. discloses the compound CHIM. This molecule is a cationic sterol derivative with a carbamoyl linker group and an imidazole as base but only one spacer and no second linker group.

WO 97/39019 of Deshmukh et al. describes different cationic sterol compounds with a carbamoyl linker group and cyclic N containing bases, such as morpholine. However, these compounds have only one spacer and no second linker group.

Objects of the invention

The object of the present invention was to provide additional, alternative sterol derivative of the general formula 1 having improved stability upon storage.

Another object of the present is the provision of liposomes comprising such sterol derivatives.

Yet another object of the present invention is to provide liposomes comprising such sterol derivatives and encapsulating an active agent.

Yet another object of the invention is to provide liposomes comprising such sterol derivatives and allow effective transfection of cells.

Yet another object of the invention is to provide pharmaceutical compositions comprising such liposomes as carriers for the delivery of active agents or ingredients.

Summary of the invention

The object of the present invention is accomplished by the technical features of claims 1 , 7, 17, 18 and 19.

Provided are facultative cationic sterol of general formula 1

cation - spacer 2 - Y - spacer 1 - X - steryl, (1)

wherein the cation is a nitrogen base with a pKa value of between 3.5 and 8, the spacers 1 is a lower alkyl residue of linear, branched or cyclic structure, which has from 1 to 8 C atoms and includes O ₁ 1 or 2 or 3 ethylenically unsaturated bonds, the spacer 2 is a lower alkyl residue of linear, branched or cyclic structure, which has from 0 to 8 C atoms and includes 0, 1 or 2 or 3 ethylenically unsaturated bonds, at least one of the linking group X or Y has the structure -NH-(C=O)-O- or -0-(C=O)- NH- and the other linking group X or Y a structure selected from -(C=O)-NH-; -O- (C=O)-NH-; -NH-(C=O)-O-; -(C=O)-S-; -O-; -NH-; -S-; -CH=N-.-O-(O=C)-; -(C=O)-O- ; -S-(O=C)-; -NH-(O=C)- Or -N=CH-, the overall molecule has a pka value of between 3.5 and 8.

Surprisingly the inventors have found that sterol derivatives comprising the above mentioned linking groups X and/or Y show an improved stability. Therefore liposomes, comprising the novel lipids can be stored in suspension for extended periods of time.

The steryls may be selected from the group consisting of cholesteryl, sitosteryl, campesteryl, desmosteryl, fucosteryl, 22-ketosteryl, 20-hydroxysteryl, stigmasteryl, 22-hydroxycholesteryl, 25-hydroxycholesteryl, lanosteryl, 7-dehydrocholesteryl, dihydrocholesteryl, 19-hydroxycholesteryl, 5α-cholest-7-en-3β-yl, 7-hydroxy- cholesteryl, epicholesteryl, ergosteryl, dehydroergosteryl and derivatives thereof.

The nitrogen base may be selected from unsubstituted or substituted piperazines, imidazoles, morpholines, purines, pyrimidines and /or pyridines or may derive from single or multiple substituted nitrogen atoms with lower hydroxyalkyls.

In one embodiment of the invention the inventive sterol derivatives have a pka value of between 5 and 8.

In another embodiment of the invention the pka value of inventive sterol derivatives may be between 4 and 7.

Another aspect of the present invention relates to liposomes comprising the inventive cationic sterol derivatives.

The liposomes may further comprise one or more neutral and/or zwitterionic lipids and/or one or more anionic lipids and/or one or more cationic lipids.

In a particular embodiment of the present invention the liposomes comprising the inventive cationic sterols are amphoteric liposomes.

Said amphoteric liposomes may comprise an anionic lipid selected from the group consisiting of DOGS, DMGS, Chol-C1 , Chol-C2, Chol-C3, Chems, Chol-C5, Chol-C6, Chol-C7 and/or Chol-C8.

The liposomes according to the invention may further comprise neutral or zwitterionic lipids, preferably selected from POPC, DPPC, DSPC, DOPC, DMPC, POPE, DPPE, DSPE, DMPE, DOPE, DPhyPE, DLinPE or natural equivalents thereof or cholesterol.

The abbreviations for the lipids used herein can be found below in this disclosure.

In one embodiment said neutral and/or zwitterionic lipid is cholesterol or a mixture of phosphatidylethanolamine and cholesterol or a mixture of phosphatidylcholine and cholesterol.

In another embodiment said neutral and/or zwitterionic lipid is a mixture of phosphatidylethanolamine and phosphatidylcholine.

In one embodiment the size of the liposomes is of between 20 to 1000 nm.

In specific embodiments of the invention the liposomes comprise an active agent.

In a preferred embodiment the active agent is a nucleic acid drug, such as oligonucleotides or plasmids.

The inventive liposomes may be used as a vector for in vivo, in vitro or ex vivo transfection.

Furthermore the liposomes may be useful for intravenous, subcutaneous, peritoneal, local or topical application.

Another aspect of the present invention relates to pharmaceutical compositions comprising the inventive liposomes for the treatment or prophylaxis of inflammatory, immune or autoimmune disorders, cancer and/or metabolic diseases of humans or non-human animals.

Detailed description of the invention

Lipids according to the invention

The present invention relates to novel facultative cationic sterol derivatives of the general formula 1 :

Cation - Spacer 2 - Y - Spacer 1 - X - Sterol (1 ).

wherein the cation is a nitrogen base with a pKa value of between 3.5 and 8, the spacers 1 is a lower alkyl residue of linear, branched or cyclic structure, which has from 1 to 8 C atoms and includes 0, 1 or 2 or 3 ethylenically unsaturated bonds, the spacer 2 is a lower alkyl residue of linear, branched or cyclic structure, which has from 0 to 8 C atoms and includes 0, 1 or 2 or 3 ethylenically unsaturated bonds, at least one of the linking group X or Y has the structure -NH-(C=O)-O- or -O-(C=O)- NH- and the other linking group X or Y a structure selected from -(C=O)-NH-; -O- (C=O)-NH-; -NH-(C=O)-O-; -(C=O)-S-; -O-; -NH-; -S-; -CH=N-.-O-(O=C)-; -(C=O)-O- ; -S-(O=C)-; -NH-(O=C)- Or -N=CH-,

the steryls may be selected from the group consisting of cholesteryl, sitosteryl, campesteryl, desmosteryl, fucosteryl, 22-ketosteryl, 20-hydroxystery!, stigmasteryl, 22-hydroxycholesteryl, 25-hydroxycholesteryl, lanosteryl, 7-dehydrocholesteryl, dihydrocholesteryl, 19-hydroxycholesteryl, 5α-cholest-7-en-3β-yl, 7-hydroxy- cholesteryl, epicholesteryl, ergosteryl, dehydroergosteryl and derivatives thereof, the overall molecule has a pka value of between 3.5 and 8.

The overall molecule assumes its pH-dependent charge characteristics by one or more nitrogen bases with a pKa value between 3.5 and 8. These nitrogen bases are linked to the 3-position of the sterol skeleton via spacers and coupling groups, thus forming a compound according to the formula of the invention. In many cases, e.g. where the nitrogen bases are in the form of a ring system, positional isomers are existing, wherein the linking spacer is substituted to various positions of the organic cation. Such positional isomers fall within the disclosure of this invention. In many cases, the pKa values of the organic cation can be influenced via said positional isomerism alone. The relevant fundamental rules are well-known to those skilled in the art. Alternatively, these effects can be estimated from tabular compilations

(Handbook of Chemistry and Physics, Vol. 73, pp. 8-37ff.).

Preferred pka values of the inventive facultative cationic sterol derivatives may depend on the appropriate application of the lipid.

Therefore, in one embodiment of the invention the pka value of the sterol derivative may be between 5 and 8.

In another embodiment of the invention, the sterol derivative may have a pKa value of between 4 and 7. Advantageously, these pKa values fall in a range which is of crucial importance for the physiology of numerous organisms.

In a preferred embodiment of the invention, the cations are nitrogen bases.

In one embodiment of the invention the cations may be derived from piperazines, imidazoles, morpholines, purines, pyrimidines, pyridines, piperidines, anilines, anisidines, toluidines, phenetidines, benzimidazoles, isochinolines, picolines, pterines, purines, pyrazines, pyridazines, chinolines, thiazoles or triazines, pyrazols,

imidazolines, imidazolidins, pyrazolines, pyrazolidines or hydrazins or derivatives thereof.

The cations preferably can be derived from piperazines, imidazoles, morpholines, purines, pyrimidines and/or pyridines or derivatives thereof.

Coupling reactions result in amphiphilic organic cations, e.g. those derived from the following classes of substances: o-, m-, p-anilines; 2-, 3- or 4-substituted anisidines, toluidines or phenetidines; 2-, 3-, 5-, 6-, 7- or 8-substituted benzimidazoles, 2-, 3-, 4- or 5-substituted imidazoles, 1- or 5-substituted isoquinolines, 2-, 3- or 4-substituted morpholines, 2-, 3- or 4-substituted picolines, 2-, 3- or 4-substituted piperidines, 1-, 2- or 3-substituted piperazines, 2-, 5- or 6-modified pterines, 3-, A-, 5-, 6- or 9-substituted purines, 2- or 3-substituted pyrazines, 3- or 4-substituted pyridazines, 2-, 3- or 4-modified pyridines, 2-, 4-, 5- or 6-substituted pyrimidines, 1-, 2-, 3-, 4-, 5-, 6- or 8-substituted quinolines, 2-, 4- or 5- substituted thiazoles, 2-, 4- or 6-substituted triazines, 1- ,3- ,4-or 5-substituted pyrazols, 2-, 3-, 4- or 5-substituted imidazolines, 1-, 2-, 3- or 4-substituted imidazolidines, 2-, 3- or 4-substituted pyrazolines, 1-, 2- or 3-substituted pyrazolidines or derivatives of tyrosine.

Particularly preferred are piperazines, imidazoles, morpholines, purines, pyrimidines and /or pyridines or derivatives thereof.

Most preferred as cations are imidazoles and/or morpholines or derivatives thereof.

In one embodiment of the invention molecule fragments such as occurring in biological systems are preferred, i.e., for example: 4-imidazoles (histamines), 2-, 6- or 9-purines (adenines, guanines, adenosines, or guanosines), 1-, 2- or 4-pyrimidines (uracils, thymines, cytosines, uridines, thymidines, cytidines), or pyridine-3-carboxylic acids (nicotinic esters or amides).

The above-mentioned structural fragments may also have additional substituents. For example, these can be methyl, ethyl, propyl, or isopropyl residues, more

preferably in hydroxy lated form, including one or two hydroxy! groups. Also, these can be hydroxyl or keto functions in the ring system.

Nitrogen bases with preferred pKa values are also formed by single or multiple substitution of the nitrogen atom with lower alkanehydroxyls such as hydroxymethyl or hydroxyethyl groups. Suitable organic bases from this group are e.g. aminopropanediols, thethanolamines, tris(hydroxymethyl)methylamines, bis(hydroxymethyl)methylamines, tris(hydroxyethyl)methylamines, bis(hydroxyethyl)- methylamines, or the corresponding substituted ethylamines. Coupling of these fragments to the hydrophobic portion of the molecule may proceed either via the nitrogen of the base or via any of the hydroxyl functions.

Particularly preferred are β- or γ-hydroxy substituted amines as nitrogen bases.

In addition to sterol derivatives including a single organic cation, those including two or three identical or different groups are also preferred. All of these groups are required to have a pKa value in the above-mentioned range. One suitable complex group is the amide of histamine and histidine or of histamine and histidylhistidine.

In a preferred embodiment of the invention the linking group X has the structure -NH- (C=O)-O- or -0-(C=O)-NH- and the linking group Y a structure selected from -(C=O)- NH-; -0-(C=O)-NH-; -NH-(C=O)-O-; -(C=O)-S-; -O-; -NH-; -S-; -CH=N-.-O-(O=C)-; - (C=O)-O-; -S-(O=C)-; -NH-(O=C)- or -N=CH-.

In another preferred embodiment of the invention the linking group X has a structure selected from -(C=O)-NH-; -0-(C=O)-NH-; -NH-(C=O)-O-; -(C=O)-S-; -O-; -NH-; -S-; -CH=N-.-O-(O=C)-; -(C=O)-O-; -S-(O=C)-; -NH-(O=C)- or -N=CH- and the linking group Y a structure -0-(C=O)-NH- or -NH-(C=O)-O-.

In another preferred embodiment of the invention, spacer 1 is a lower alkyl residue of linear, branched or cyclic structure, which has from 1 to 8 C atoms and includes O, 1 or 2 or 3 ethylenically unsaturated bonds. Spacer 1 may have hydroxyl groups so as to increase the polarity of the molecule. In particular, spacer 1 can be a sugar or sugaralcohol. Spacer 2 is a lower alkyl residue of linear, branched or cyclic structure,

which has from 0 to 8 C atoms and includes 0, 1 or 2 or 3 ethylenically unsaturated bonds. Spacer 2 may have hydroxy! groups so as to increase the polarity of the molecule. In particular, spacer 2 can be a sugar or sugaralcohol. In one embodiment spacer 2 is a lower alkyl residue of linear, branched or cyclic structure, which has from 1 to 8 C atoms and includes 0, 1 or 2 or 3 ethylenically unsaturated bonds.

Surprisingly it was found that the inventive sterol derivatives show an improved stability compared to the lipid MoChol as described in example 5.

Methods of performing such coupling reactions are well-known to those skilled in the art and may vary depending on the starting material and coupling component employed. Typical reactions are acylation, esterification, amidation, addition of amines to double bonds, etherification, or reductive amination.

In another preferred embodiment of the invention, the steryls are particularly cholesteryl, sitosteryl, campesteryl, desmosteryl, fucosteryl, 22-ketosteryl, 20- hydroxysteryl, stigmasteryl, 22-hydroxycholesteryl, 25-hydroxycholesteryl, lanosteryl, 7-dehydrocholesteryl, dihydrocholesteryl, 19-hydroxycholesteryl, 5α-cholest-7-en-3β- yl, 7-hydroxycholesteryl, epicholesteryl, ergosteryl, and/or dehydroergosteryl, as well as other related compounds.

The steryls that are used may bear various groups in the 3-position thereof, which groups allow for ready and stable coupling or optionally assume the function of a spacer. Particularly suitable for direct coupling are the hydroxyl group which is naturally present, but also, the chlorine of steryl chlorides, or e.g. the amino group of sterylamines, or the thiol group of thiocholesterol.

In one aspect facultative cationic sterols according to the present invention preferably have the following general formula 2:

wherein

Spacer 1 and Spacer 2 are as defined above and Y is selected from -(C=O)-NH-; -NH-(O=C)-; -0-(C=O)-NH-; -NH-(C=O)-O-; -O-;.-O-(O=C)- Or -(C=O)- O- and the cation is selected from substituted or unsubstituted piperazines, imidazoles, morpholines, purines, pyrimidines , pyridines and/or β- or γ-hydroxy substituted amines

In one embodiment of this aspect the inventive sterol may comprise, but not limited to, one of the following structures:

Table 1 :

Compound #1

CholC3N-Mo2

[^-Morpholine^-yl-ethylcarbamoyOmethylJ-carbamic acid cholesteryl ester

Compound #2

CholC4N-Mo2

[(2-Morpholine-4-yl-ethylcarbamoyl)-ethyl]-carbamic acid cholesteryl ester

Compound #3

CholC3N-Mo3

[(S-Morpholine^-yl-propylcarbamoyO-methyll-carbamic acid cholesteryl ester

Compound #4

CholDMC4N-Mo2

[1 -Methyl-2-(2-morpholine-4-yl-ethylcarbamoyl)-propyl]-carbami c acid cholesteryl ester

Compound #5

Compound #6

Compound #7

Compound #8

Compound #9

Compound #10

Liposomes according to the invention

The invention also relates to liposomes comprising the facultative cationic sterols according to the invention. The inventive facultative cationic sterols can be incorporated in high amounts in liposomal membranes.

In a special embodiment of the invention, the amount of the inventive sterol derivative in the liposomal membrane is between 3 and 100 mol%, preferred between 5 and 80 mol% and particularly preferred between 7 and 70 mol%.

In one embodiment of the invention the liposomes may comprise one or more of the inventive lipids and one or more neutral and/or zwitterionic lipid. Examples of such lipids include phosphatidyl choline, phosphatidyl ethanolamine, sphingomylein, ceramides, diacylglycerol and/or cholesterol.

In a preferred embodiment the phosphatidylcholine may be selected from the group comprising POPC, natural or hydrogenated soya PC, natural or hydrogenated egg PC, DMPC, DPPC, DSPC or DOPC. The phosphatidylethanolamines are preferably selected from the group comprising DOPE, DMPE or DPPE or POPE, DPhyPE, DLinPE, DSPE or natural equivalents thereof.

In another embodiment of the present invention liposomes comprising the inventive lipids may comprise one or more anionic lipids and/or one or more cationic lipids. Examples of anionic and cationic lipids are given below in this disclosure.

In a particular aspect of the invention the liposomes comprising the inventive lipids are amphoteric liposomes. Amphoteric liposomes are a new class of liposomes described in WO 02/066012. Amphoteric liposomes have an anionic or neutral charge at pH 7.5 and a cationic charge at pH 4. These liposomes can be prepared from lipid mixtures comprising either an amphoteric lipid or a mixture of lipids with amphoteric character and optional a neutral and/or zwitterionic lipid.

The amphoteric liposomes comprising the inventive sterol lipids according to the present invention can be formed from any amphoteric lipid mixture.

In one embodiment the amphoteric lipid mixture may comprise a plurality of charged amphiphiles which in combination with one another have amphoteric character. A selection of charged amphiphiles is summarized below in this disclosure. In a preferred aspect of this embodiment said one or more charged amphiphiles comprise a pH sensitive anionic lipid and a pH sensitive cationic lipid. Herein, such a combination of a chargeable cation and chargeable anion is referred to as an "amphoteric II" lipid pair. In a preferred embodiment of this aspect said chargeable cations have pKa values of between about 5 and about 8. In a preferred embodiment of this aspect said chargeable cation is a facultative cationic sterol according to the present invention.

In another aspect of this embodiment said one or more charged amphiphiles comprise a stable anion and a chargeable cation and is referred to as "amphoteric III" lipid pair. In a preferred embodiment of this aspect said chargeable cations have pKa values of between about 4 and about 7. In a preferred embodiment of this aspect said chargeable cation is a facultative cationic sterol according to the present invention.

It is of course possible to use amphiphiles with multiple charges such as amphipathic dicarboxylic acids, phosphatidic acid, amphipathic piperazine derivatives and the like. Such multicharged amphiphiles might fall into pH sensitive amphiphiles or stable anions or cations or might have mixed character.

Suitable anionic lipids for the formation of amphoteric liposomes in combination with the inventive cationic sterols include, but are not limited to diacylglycerolhemisuccinates, e.g. DOGS, DMGS, POGS, DPGS, DSGS; diacylglycerolhemimalonat.es, e.g. DOGM or DMGM; diacylglycerolhemiglutarates,

e.g. DOGG, DMGG; diacylglycerolhemiadipates, e.g. DOGA, DMGA; diacylglycerolhemicyclohexane-1 ,4-dicarboxylic acids, e.g. DO-cHA, DM-cHA; (2,3- Diacyl-propyl)amino}-oxoalkanoic acids e.g. DOAS ₁ DOAM, DOAG, DOAA, DMAS, DMAM ₁ DMAG, DMAA; Diacyl-alkanoic acids, e.g. DOP ₁ DOB, DOS, DOM ₁ DOG ₁ DOA, DMP ₁ DOB, DMS, DMM, DMG ₁ DMA; Chems and derivatives therof, e.g. Chol- C2, Chol-C3, Chol-C5 or Chol-C6, Chol-C7, Chol-C8; Chol-C1 , fatty acids, e.g. Oleic acid, Linoleic Acid, Myristic Acid, Palmitic acid, Stearic acid, Nervonic Acid, Behenic Acid; DOPA ₁ DMPA, DPPA, POPA, DSPA, Chol-SO4, DOPG, DMPG, DPPG, POPG, DSPG or DOPS ₁ DMPS ₁ DPPS, POPS, DSPS, Cetyl-phosphate or any of compounds 1-22 of table 3 below.

Any dialkyl derivatives of these anionic lipids comprising diacyl groups are also within the scope of the present invention.

Preferred anionic lipids for the formation of amphoteric liposomes in combination with the inventive cationic sterols may be selected from the group consisting of DOGS, DMGS, Chol-CI , Chol-C2, Chol-C3, Chems, Chol-C5, Chol-C6, Chol-C7 and/or Chol-C8.

In some embodiments of the invention the amphoteric lipid mixtures further comprise one or more neutral and/or zwitterionic lipids. Examples of such lipids may include phosphatidyl choline, phosphatidyl ethanolamine, sphingomylein, ceramides, diacylglycerol and/or cholesterol.

In one aspect of this embodiment said neutral lipids may be cholesterol or a mixture of cholesterol and phosphatidylethanolamines. Preferably said lipids are present in the amphoteric liposomes as sole neutral lipids.

Preferably the molar ratio of the mixtures of cholesterol and phosphatidylethanolamine is between 4 and 0.25, preferred between 3 and 0.5 and most preferred between 2 and 1.

Examples of phosphatidylethanolamines may include POPE, DPPE, DSPE, DMPE, DOPE, DPhyPE, DLJnPE or natural equivalents thereof.

Mixtures of different phosphatidylethanolamines are also within the scope of the present invention. In a preferred embodiment of the invention the phosphatidylethanolamine is DOPE.

In another aspect of this embodiment a mixture of neutral lipids, such as phosphatidylcholines (PC), sphingomyelins or ceramides and cholesterol (Choi) may be used as sole neutral lipid components in the amphoteric liposomes.

Preferred are mixtures of phosphatidylcholines and cholesterol. The molar ratio of PC/Choi may be between 4 and 0.25 or between 3 and 0.33. Preferred are molar ratios of PC/Choi between 1.5 and 0,25, more preferred between 1 and 0.25.

The phosphatidylcholines may be selected without limitation from the group POPC, DOPC, DMPC, DPPC, DSPC or natural equivalents thereof, such as soy bean PC or egg-PC. Mixtures of different phosphatidylcholines are also within the scope of the present invention. In a preferred embodiment of the invention the phosphatidylcholine is selected from POPC or DOPC.

In a still further aspect of this embodiment a mixture of phosphatidylcholines, sphingomyelins or ceramides and phosphatidylethanolamines may be used as sole neutral lipid components in the amphoteric liposomes. Preferred are mixtures of phosphatidylcholines and phosphatidylethanolamines. In a preferred embodiment of the invention the phosphatidylcholine is POPC and the phosphatidylethanolamine is DOPE.

Obviously, further modifications of the liposomes according to the present invention are possible. Thus, the use of polyethylene glycol-modified phospholipids or analogous products is particularly advantageous. Other examples of lipids or compounds which sterically stabilize liposomes and thereby avoid an uptake of the particles by the RES (reticuloendothelial system) upon injection of the particles into the blood stream include for example polyglycerols, dextranes, polysialic acids, hydroxyethyl starches, hyaluronic acids, PEGylated lipids, sugar alcohols, Tween 80 or GM 1 gangliosides. Such lipids or detergents are known in the art to sterically

stabilize liposomes (e.g. Woodle et al., Biochim. Biophys. Acta, 1113(2), 171-179, (1992); Allen et al., Biochim. Biophys. Acta, 981(1), 27-35, (1989)).

In one embodiment of the invention, the liposomes or amphoteric liposomes according to the present invention have an average size of between 50 and 1000 nm, preferably between 50 and 300 nm, and more preferably between 60 and 130 nm. In another embodiment the size of the liposomes or amphoteric liposomes may be less than 50nm, preferably of between 20 and 50 nm.

In one embodiment of the invention amphoteric liposomes comprising the inventive cationic sterol derivatives may be selected from one of the following formulations:

Table 2:

In another preferred embodiment, the liposomes comprise active substances. For example, the liposomes according to the invention are suitable for parenteral application. They can be used e.g. in cancer therapy and in the therapy of severe infections. To this end, liposome dispersions can be injected, infused or implanted. Thereafter, they are distributed in the blood or lymph or release their active substance in a controlled fashion as a depot. The latter can be achieved by highly concentrated dispersions in the form of gels. The liposomes can also be used for

topical application on the skin. In particular, they may contribute to improved penetration of various active substances into the skin or even passage through the skin and into the body. Furthermore, the liposomes can also be used in gene transfer. Due to its size and charge, genetic material is usually incapable of entering cells without an aid. For this purpose, suitable carriers such as liposomes or lipid complexes are required which, together with the DNA, are to be taken up by the respective cells in an efficient and well-directed fashion. To this end, cell-inherent transport mechanisms such as endocytosis are used. Obviously, the liposomes of the invention can also be used as model membranes. In their principal structure, liposomes are highly similar to cell membranes. Therefore, they can be used as membrane models to quantify the permeation rate of active substances through membranes or the membrane binding of active substances.

Without being limited to such use, the liposomes in the present invention are well suited for use as carriers for active ingredients such as for example proteins, peptides, nucleic acids.

In one embodiment of the invention the active ingredient is a nucleic acid.

These drugs are classified into nucleic acids that encode one or more specific sequences for proteins, polypeptides or RNAs and into oligonucleotides that can specifically regulate protein expression levels or affect the protein structure through inter alia interference with splicing and artificial truncation. In some embodiments of the present invention, therefore, the nucleic acid-based therapeutic may comprise a nucleic acid that is capable of being transcribed in a vertebrate cell into one or more RNAs, which RNAs may be mRNAs, shRNAs, miRNAs or ribozymes, wherein such mRNAs code for one or more proteins or polypeptides. Such nucleic acid therapeutics may be circular DNA plasmids, linear DNA constructs, like MIDGE vectors (Minimalistic lmmunogenically Defined Gene Expression) as disclosed in WO 98/21322 or DE 19753182, or mRNAs ready for translation (e.g., EP 1392341).

In another embodiment of the invention, oligonucleotides may be used that can target existing intracellular nucleic acids or proteins. Said nucleic acids may code for a specific gene, such that said oligonucleotide is adapted to attenuate or modulate

transcription, modify the processing of the transcript or otherwise interfere with the expression of the protein. The term "target nucleic acid" encompasses DNA encoding a specific gene, as well as all RNAs derived from such DNA, being pre-mRNA or mRNA. A specific hybridisation between the target nucleic acid and one or more oligonucleotides directed against such sequences may result in an inhibition or modulation of protein expression. To achieve such specific targeting, the oligonucleotide should suitably comprise a continuous stretch of nucleotides that is substantially complementary to the sequence of the target nucleic acid. Oligonucleotides fulfilling the abovementioned criteria may be built with a number of different chemistries and topologies. The oligonucleotides may comprise naturally occurring or modified nucleosides comprising but not limited to DNA, RNA, locked nucleic acids (LNA's), 2'O-methyl RNA (2'Ome), 2' O-methoxyethyl RNA (2 ¹ MOE) in their phosphate or phosphothioate forms or Morpholinos or peptide nucleic acids (PNA ¹ S). Oligonucleotides may be single stranded or double stranded.

Oligonucleotides are polyanionic structures having 8-60 charges. In most cases these structures are polymers comprising nucleotides. The present invention is not limited to a particular mechanism of action of the oligonucleotides and an understanding of the mechanism is not necessary to practice the present invention. The mechanisms of action of oligonucleotides may vary and might comprise inter alia effects on splicing, transcription, nuclear-cytoplasmic transport and translation. In a preferred embodiment of the invention single stranded oligonucleotides may be used, including, but not limited tθτ DNA-based oligonucleotides, locked nucleic acids, 2'-modified oligonucleotides and others, commonly known as antisense oligonucleotides. Backbone or base or sugar modifications may include, but are not limited to, Phosphothioate DNA (PTO), 2'O-methyl RNA (2'Ome), 2'Fluoro RNA (2'F), 2' O- methoxyethyl-RNA (2'MOE), peptide nucleic acids (PNA), N3"-P5' phosphoamidates (NP) ₁ 2'fluoroarabino nucleic acids (FANA), locked nucleic acids (LNA), Morpholine phosphoamidate (Morpholino), Cyclohexene nucleic acid (CeNA), tricyclo-DNA (tcDNA) and others. Moreover, mixed chemistries are known in the art, being constructed from more than a single nucleotide species as copolymers, block- copolymers or gapmers or in other arrangements.

In addition to the aforementioned oligonucleotides, protein expression can also be inhibited using double stranded RNA molecules containing the complementary

sequence motifs. Such RNA molecules are known as siRNA molecules in the art (e.g., WO 99/32619 or WO 02/055693). Other siRNAs comprise single stranded siRNAs or double stranded siRNAs having one non-continuous strand. Again, various chemistries were adapted to this class of oligonucleotides. Also, DNA / RNA hybrid systems are known in the art.

In another embodiment of the present invention, decoy oligonucleotides can be used. These double stranded DNA molecules and chemical modifications thereof do not target nucleic acids but transcription factors. This means that decoy oligonucleotides bind sequence-specific DNA-binding proteins and interfere with the transcription (e.g., Cho-Chung, et al. in Curr. Opin. MoI. Then, 1999).

In a further embodiment of the invention oligonucleotides that may influence transcription by hybridizing under physiological conditions to the promoter region of a gene may be used. Again various chemistries may adapt to this class of oligonucleotides. In a still further alternative of the invention, DNAzymes may be used. DNAzymes are single-stranded oligonucleotides and chemical modifications therof with enzymatic activity. Typical DNAzymes, known as the "10-23" model, are capable of cleaving single-stranded RNA at specific sites under physiological conditions. The 10-23 model of DNAzymes has a catalytic domain of 15 highly conserved deoxyribonucleotides, flanked by 2 substrate-recognition domains complementary to a target sequence on the RNA. Cleavage of the target mRNAs may result in their destruction and the DNAzymes recycle and cleave multiple substrates.

In yet another embodiment of the invention, ribozymes can be used. Ribozymes are single-stranded oligoribonucleotides and chemical modifications thereof with enzymatic activity. They can be operationally divided into two components, a conserved stem-loop structure forming the catalytic core and flanking sequences which are reverse complementary to sequences surrounding the target site in a given

RNA transcript. Flanking sequences may confer specificity and may generally constitute 14-16 nt in total, extending on both sides of the target site selected.

In a still further embodiment of the invention _τ aptamers may be used to target proteins. Aptamers are macromolecules composed of nucleic acids, such as RNA or DNA, and chemical modifications thereof that bind tightly to a specific molecular

target and are typically 15-60 nt long. The chain of nucleotides may form intramolecular interactions that fold the molecule into a complex three-dimensional shape. The shape of the aptamer allows it to bind tightly against the surface of its target molecule including but not limited to acidic proteins, basic proteins, membrane proteins, transcription factors and enzymes. Binding of aptamer molecules may influence the function of a target molecule.

All of the above-mentioned oligonucleotides may vary in length between as little as 5 or 10, preferably 15 and even more preferably 18, and 50, preferably 30 and more preferably 25, nucleotides per strand. More specifically, the oligonucleotides may be antisense oligonucleotides of 8 to 50 nucleotides length that catalyze RNAseH mediated degradation of their target sequence or block translation or re-direct splicing or act as antogomirs; they may be siRNAs of 15 to 30 basepairs length; they may further represent decoy oligonucleotides of 15 to 30 basepairs length; can be complementary oligonucleotides influencing the transcription of genomic DNA of 15 to 30 nucleotides length; they might further represent DNAzymes of 25 to 50 nucleotides length or hbozymes of 25 to 50 nucleotides length or aptamers of 15 to 60 nucleotides length. Such subclasses of oligonucleotides are often functionally defined and can be identical or different or share some, but not all features of their chemical nature or architecture without substantially affecting the teachings of this invention. The fit between the oligonucleotide and the target sequence is preferably perfect with each base of the oligonucleotide forming a base pair with its complementary base on the target nucleic acid over a continuous stretch of the abovementioned number of oligonucleotides. The pair of sequences may contain one or more mismatches within the said continuous stretch of base pairs, although this is less preferred. In general the type and chemical composition of such nucleic acids is of little impact for the performance of the inventive liposomes as vehicles be it in vivo or in vitro and the skilled artisan may find other types of oligonucleotides or nucleic acids suitable for combination with the inventive liposomes.

Advantageously, liposomes produced using the sterol derivatives of the invention show low non-specific binding to cell surfaces. It is this low non-specific binding which is an essential precondition for achieving specific binding to target cells. Target control of the vehicles is obtained when providing the above-described liposomes

with additional ligands. As a result, the active substance can be accumulated specifically in such cells or tissues which exhibit a pathological condition.

One important use of the facultative cationic sterols according to the invention is therefore in the construction of vectors for transfer of active substances in living organisms. The vectors are particularly suited for the transport of therapeutic macromolecules such as proteins or DNA which themselves are incapable of penetrating the cell membrane or undergo rapid degradation in the bloodstream.

Advantageously, antibodies, lectins, hormones or other active substances can be coupled to the surface of liposomes under mild conditions in high yields.

In a particularly preferred embodiment of the invention, at least 80% of the active substance is inside the liposome.

In another embodiments of the invention the liposomes comprise non-encapsulated active agents.

The preparation of liposomes is well known in the art (e.g. Liposomes, Ed. V.P.

Torchilin and V. Weissig, Second Edition, Oxford University Press, USA). Specific examples for the preparation of liposomes include, but are not limited to, the lipid film/hydration procedure with subsequent optional extrusion or sonication or the injection of an ethanolic lipid solution in water or buffer.

Amphoteric liposomes offer the distinct advantage that they can encapsulate nucleic acids with high efficiencies as disclosed for example in WO 02/066012 or WO 07/107304 both of Panzner et al..

The invention also relates to the use of the liposomes in the production of nanocapsules.

The invention also relates to the use of the liposomes in the production of release systems in diagnostics.

Advantageously, the liposomes are used for the transport and/or release of active substances.

In another embodiment, the liposomes conveniently are used as depot formulation and/or as circulative depot.

Advantageously, the liposomes can be used in intravenous or peritoneal application.

In another embodiment of the invention, the liposomes are used with advantage as vector to transfect cells in vivo, in vitro and ex vivo.

A further aspect of the invention relates to pharmaceutical compositions comprising liposomes comprising the cationic sterol derivatives according to the present invention preferably as a carrier for the delivery of active agents or ingredients, including drugs such as nucleic acid drugs, e.g., oligonucleotides and plasmids. The pharmaceutical composition of the present invention may be formulated in a suitable pharmacologically acceptable vehicle. Vehicles such as water, saline, phosphate buffered saline, D5W and the like are well known to those skilled in the art for this purpose. In some embodiments the pharmaceutical compositions according to the invention may be used particularly for the treatment or prophylaxis of inflammatory, immune or autoimmune disorders, cancer and/or metabolic diseases of humans or non-human animals. A yet further aspect of the present invention relates to methods for the treatment of human or non-human animals in which said pharmaceutical composition comprising the inventive liposomes or amphoteric liposomes are targeted to a specific organ or organs, tumours or sites of infection or inflammation.

Definitions

By "chargeable" is meant that the amphiphile has a pK in the range pH 4 to pH 8. A chargeable amphiphile may therefore be a weak acid or base. A "stable" amphiphile is a strong acid or base, having a substantially stable charge on the range pH 4 to pH 8.

By "amphoteric" herein is meant a substance, a mixture of substances or a supra- molecular complex (e.g., a liposome) comprising charged groups of both anionic and cationic character wherein:

1) at least one, and optionally both, of the cation and anionic amphiphiles is chargeable, having at least one charged group with a pK between 4 and 8,

2) the cationic charge prevails at pH 4, and

3) the anionic charge prevails at pH 8.

As a result the substance or mixture of substances has an isoelectric point of neutral net charge between pH 4 and pH 8. Amphoteric character is by this definition different from zwittehonic character, as zwitterions do not have a pK in the range mentioned above. In consequence, zwitterions are essentially neutrally charged over a range of pH values; phosphatidylcholines and phosphatidylethanolamines are neutral lipids with zwitterionic character.

By "C/A" or "C/A ratio" or "CVA molar ratio" herein is meant the molar ratio of cationic amphiphiles to anionic amphiphiles in a mixture of amphiphiles.

By "IC50" herein is meant the inhibitory concentration of an oligonucleotide leading to a 50 % knockdown of a target mRNA or in case of a proliferation assay to a 50 % inhibition of cell viability.

By "facultative cationic" herein is meant that the charge of a cation or a base depends on the pH of the medium.

The following list of lipids includes specific examples of neutral, zwitterionic, anionic, cationic or amphoteric lipids mentioned in this disclosure. The lipid list by no means limits the scope of this disclosure. The abbreviations for the lipids are used herein, the majority of which abbreviations are in standard use in the literature:

Neutral or zwitterionic lipids:

PC Phosphatidylcholine (unspecified membrane anchor)

PE Phosphatidylethanolamine (unspecified membrane anchor)

SM Sphingomyelin (unspecified membrane anchor)

DMPC Dimyristoylphosphatidylcholine

DPPC Dipalmitoylphosphatidylcholine

DSPC Distearoylphosphatidylcholine

POPC 1 -Palmitoyl^-oleoylphosphatidylcholine

DOPC Dioleoylphosphatidylcholine

DOPE Dioleoylphosphatidylethanolamine DMPE Dimyristoylphosphatidylethanolamine

DPPE Dipalmitoylphosphatidylethanolamine

DPhyPE Diphytanoylphosphatidylethanolamine

DlinPE Dilinoleoylphosphatidylethanolamine

Choi Cholesterol

Any dialkyl derivatives of the neutral or zwitterionic lipids comprising diacyl groups listed above are also within the scope of the present invention.

Anionic lipids:

CHEMS Cholesterolhemisuccinate

Chol-COOH or Chol-C1 Cholesteryl-3-carboxylic acid

Chol-C2 Cholesterolhemioxalate

Chol-C3 Cholesterolhemimalonate Chol-C5 Cholesterolhemiglutarate

Chol-C6 Cholesterolhemiadipate

Chol-C7 Cholesterolhemipimelate

Chol-C8 Cholesterolhemisuberate

DGS or DG-Succ Diacylglycerolhemisuccinate (unspecified membrane anchor) DOGS or DOG-Succ Dioleoylglycerolhemisuccinate

DMGS or DMG-Succ Dimyristoylglycerolhemisuccinate

DPGS or DPG-Succ Dipalmitoylglycerolhemisuccinate

DSGS or DSG-Succ Distearoylglycerolhemisuccinate POGS or POG-Succ i-Palmitoyl-2-oleoylglycerolhemisuccinate

DOGM Dioleoylglycerolhemimalonate

DOGG Dioleoylglycerolhemiglutarate

DOGA Dioleoylglycerolhemiadipate

DMGM Dimyristoylglycerolhemimalonate

DMGG Dimyristoylglycerolhemiglutarate

DMGA Dimyristoylglycerolhemiadipate

DOAS 4-{(2,3-Dioleoyl-propyl)amino}-4-oxobutanoic acid

DOAM 3-{(2,3-Dioleoyl-propyl)amino}-3-oxopropanoic acid DOAG 5-{(2,3-Dioleoyl-propyl)amino}-5-oxopentanoic acid

DOAA 6-{(2,3-Dioleoyl-propyl)amino}-6-oxohexanoic acid

DMAS 4-{(2,3-Dimyristoyl-propyl)amino}-4-oxobutanoic acid

DMAM 3-{(2,3-Dimyristoyl-propyl)amino}-3-oxopropanoic acid

DMAG 5-{(2,3-Dimyristoyl-propyl)amino}-5-oxopentanoic acid DMAA 6-{(2,3-Dimyristoyl-propyl)amino}-6-oxohexanoic acid

DOP 2,3-Dioleoyl-propanoic acid

DOB 3,4-Dioleoyl-butanoic acid

DOS 5,6-Dioleoyl-hexanoic acid

DOM 4,5-Dioleoyl-pentanoic acid DOG 6,7-Dioleoyl-heptanoic acid

DOA 7,8-Dioleoyl-octanoic acid

DMP 2,3-Dimyristoyl-propanoic acid

DMB 3,4-Dimyristoyl-butanoic acid

DMS 5,6-Dimyristoyl-hexanoic acid DMM 4,5-Dimyristoyl-pentanoic acid

DMG 6,7-Dimyristoyl-heptanoic acid

DMA 7,8-Dimyristoyl-octanoic acid

DOG-GIuA Dioleoylglycerol-glucoronic acid (1- or 4-linked)

DMG-GIuA Dimyristoylglycerol-glucoronic acid (1- or 4-linked) DO-cHA Dioleoylglycerolhemicyclohexane-1 ,4-dicarboxylic acid

DM-cHA Dimyristoylglycerolhemicyclohexane-1 ,4-dicarboxylic acid

DOPS Dioleoylphosphatidylserine

DPPS Dipalmitoylphosphatidylserine

DOPG Dioleoylphosphatidylglycerol DPPG Dipalmitoylphosphatidylglycerol

Chol-SO4 Cholesterol sulphate

DOPA Dioleoylphosphatidic acid

SDS Sodium dodecyl sulphate

Cet-P Cetylphosphate

MA Myristic Acid

PA Palmitic Acid

OA Oleic Acid

LA Linoleic Acid

SA Stearic Acid

NA Nervonic Acid

BA Behenic Acid

Table 3:

The diacylglycerols may be dimyristoyl-, dipalmitoyl-, dioleoyl-, distearoyl- or palmitoyloleoylglycerols and R in the table above includes any selection from this group or cholesterol.

Any dialkyl derivatives of the anionic lipids comprising diacyl groups listed above are also within the scope of the present invention.

Cationic lipids:

MoChol 4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate

HisChol Histaminyl-Cholesterolhemisuccinate

CHIM Cholesterol-(3-imidazol-1-yl propyl)carbamate

DmC4Mo2 4-(2-Aminoethyl)-Morpholino-Cholesterol-2,3-dimethylhemisucc inate

DmC3Mo2 4-(2-Aminoethyl)-Morpholino-Cholesterol-2,2-dimethylhemimalo nate

C3Mo2 4-(2-Aminoethyl)-Morpholino-Cholesterol-hemimalonate

C3Mo3 4-(2-Aminopropyl)-Moφholino-Cholesterol-hemimalonate

C4Mo4 4-(2-Aminobutyl)-Morpholino-Cholesterol-hemisuccinate

C5Mo2 4-(2-Aminoethyl)-Morpholino-Cholesterol- hemiglutarate

C6Mo2 4-(2-Aminoethyl)-Morpholino-Cholesterol- hemiadipate

C8Mo2 4-(2-Aminoethyl)-Morpholino-Cholesterol- hemiadipate

DDAB Dimethyldioctadecylammonium bromide

1 ,2-Diacyl-3-Trimethylammonium-Propane e.g.

DOTAP 1 ,2-Dioleoyl-3-Trimethylammonium-Propane

DMTAP 1 ,2-Dimyristoyl-3-Trimethylammonium-Propane

DPTAP 1 ,2-Dipalmitoyl-3-Trimethylammonium-Propane

DSTAP 1 ,2-Distearoyl-3-Trimethylammonium-Propane

POTAP Palmitoyloleoyl-3-Trimethylammonium-Propane

1 ,2-Diacyl-3-Dimethylhydroxyethylammonium-Propane e.g.

DODMHEAP 1 ,2-Dioleoyl-3-dimethylhydroxyethylammonium-Propane

DMDMHEξAP 1 ,2-Dimyristoyl-3-dimethylhydroxyethylammonium-Propane

DPDMHEAP 1 ,2-Dipalmitoyl-3-dimethylhydroxyethylammonium-Propane DSDMHEAP 1 ,2-Distearoyl-3-dimethylhydroxyethylammonium-Propane

PODMHEAP Palmitoyloleoyl-3-dimethylhydroxyethyl-ammonium-Propane

1 ,2-Diacyl-3-methyldihydroxyethylammonium-Propane e.g.

DOMDHEAP 1 ,2-Dioleoyl-3-methyldihydroxyethylammonium-Propane DMMDHEAP 1 ,2-Dimyristoyl-3-methyldihydroxyethylammonium-Propane

DPMDHEAP 1 ,2-Dipalmitoyl-3-methyldihydroxyethylammonium-Propane

DSMDHEAP 1 ,2-Distearoyl-3-methyldihydroxyethylammonium-Propane POMDHEAP Palmitoyloleoyl-3-methyldihydroxyethyl-ammonium-Propane

1 ,2-Diacyl-3-Dimethylammonium-Propane e.g.

DODAP 1 ,2-Dioleoyl-3-Dimethylammonium-Propane DMDAP 1 ,2-Dimyristoyl-3-Dimethylammonium-PiOpane

DPDAP 1 ,2-Dipalmitoyl-3-Dimethylammonium-Propane

DSDAP 1 ,2-Distearoyl-3-Dimethylammonium-Propane

PODAP Palmitoyloleoyl-3-Dimethylammonium-Propane

1 ,2-Diacyl-3-methylhydroxyethylammonium-Propane e g-

DOMHEAP 1 ,2-Dioleoyl-3-methylhydroxyethylammonium-Propane

DMMHEAP 1 ,2-Dimyristoyl-3-methylhydroxyethylammonium-Propane

DPMHEAP 1 ,2-Dipalmitoyl-3-methylhydroxyethylammonium-Propane

DSMHEAP 1 ,2-Distearoyl-3-methylhydroxyethy!ammonium-Propane

POMHEAP Palmitoyloleoyl-3-methylhydrόxyethylammonium-Propane

1 ,2-Diacyl-3-dihydroxyethylammonium-Propane e.g.

DODHEAP 1 ,2-Dioleoyl-3-dihydroxyethylammonium-Propane

DMDHEAP 1 ,2-Dimyristoyl-3-dihydroxyethylammonium-Propane

DPDHEAP 1 ,2-Dipalmitoyl-3-dihydroxyethylammonium-Propane

DSDHEAP 1 ,2-Distearoyl-3-dihydroxyethylammonium-Propane PODHEAP Palmitoyloleoyl-3-dihydroxyethylammonium-Propane

i ^-Diacyl-sn-Glycero-S-Ethylphosphocholine e.g.

DOEPC 1 ^-Dioleoyl-sn-Glycero-S-Ethylphosphocholine DMEPC i ^-Dimyristoyl-sn-Glycero-S-Ethylphosphocholine

DPEPC 1 ^-Dipalmitoyl-sn-Glycero-S-Ethylphosphocholine

DSEPC 1 ,2-Distearoyl-sn-Glycero-3-Ethylphosphocholine

POEPC Palmitoyloleoyl-sn-Glycero-S-Ethylphosphocholine

DOTMA N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride

DOTIM 1 -[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl) imidazolinium chloride

TMAG N-(a-trimethylammonioacetyl)-didodecyl-D-glutamate chloride

BCAT O-(2R-1 ,2-di-O-(1 θZ.θθZ-octadecadienyO-glyceroO-N- (bis-2-aminoethyl)carbamate

DODAC Dioleyldimethylammonium chloride

DORIE 1 ,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide

DMRIE 1 ,2-dimyristoyl-3-dimethyl-hydroxyethyl ammonium bromide

DOSC 1 ^-dioleoyl-S-succinyl-sn-glycerol choline ester DORI 1 ,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride

DHMHAC N.N-di-n-hexadecyl-N.N-dihydroxyethylammoniumbromide

DHDEAB N,N-di-n-hexadecyl-N-methyl,N-(2- hydroxyethyl)ammonium chloride

DMHMAC N _> N-myristyl-N-(1-hydroxyprop-2-yl)-N-methylammonium chloride

DOTB 1 ,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn- glycerol DOSPA 2,3-Dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl -1- propanaminium trifluoroacetate

DOGS ^* Dioctadecylamido-glycylspermine DOGSDSO 1 ^-dioleoyl-sn-glycero-S-succinyl^-hydroxyethyl disulfide ornithine SAINT lipids Synthetic Amphiphiles INTerdisciplinary DPIM ₁ DOIM 4,(2,3-bis-acyloxy-propyl)-1-methyl-1 H- imidazole (unspecified membrane anchor)

MoDP 1 ,2-Dipalmitoyl-3-N-morpholine-propane

MoDO 1 ,2-Dioleoyl-3-N-morpholine-propane

DPAPy 2,3-bis-palmitoyl-propyl-pyhdin-4-yl-amine

DC-Choi 3b-[N-(N9,N9-dimethylaminoethane)carbamoyl] cholesterol TC-Chol 3b-[N-(N9,N9-trimethylaminoethane) carbamoyl] cholesterol

DAC-Chol 3b(N-(N,N'-Dimethylaminoethan)- carbamoyl)cholesterol

PipC2Chol 4{N-2-ethylamino[(3'-β-cholesteryl) carbamoyl]} piperazine

MoC2Chol {N-2-ethylamino[(3'-β-cholesteryl) carbamoyl]} morpholine

MoC3Chol {N-2-propylamino[(3'-β-cholesteryl) carbamoyl]} morpholine N-methyl-PipChol N-methyl{4-N-amino[(3'-β-cholesteryl) carbamoyl]}piperazine

PyrroC2Chol {N-2-ethylamino[(3'-β-cholesteryl) carbamoyl]}pyrrolidine

PipeC2Chol {N-2-ethylamino[(3'-β-cholesteryl) carbamoyl]}piperidine lmC3Chol {N-2-propylamino[(3'-β-cholesteryl) carbamoyl]}imidazole

PyC2Chol {N-2-ethylamino[(3'-β-cholesteryl) carbamoyl]}pyridine CTAB Cetyltrimethylammonium bromide

NeoPhectin™ cationic cardiolipins (e.g. [1 , 3-Bis-(1 , 2-bis-tetradecyloxy- propyl-3-dimethylethoxyammonium bromide)-propane-2-ol]

Table 4:

The diacylglycerols may be dimyristoyl-, dipalmitoyl-, dioleoyl-, distearoyl- or palmitoyloleoylglycerols and R in the table above includes any selection from this group or R may be a C ₆ -C ₃₀ dialkyl, with 0-3 unsaturated bonds.

Any dialkyl derivatives of the cationic lipids comprising diacyl groups listed above are also within the scope of the present invention.

Amphoteric lipids:

HistChol Nα-Histidinyl-Cholesterol-hemisuccinate

HistDG 1 ,2 — Dipalmitoylglycerol-hemisuccinat-N_-Histidinyl- hemisuccinate, & Distearoyl- .Dimyristoyl, Dioleoyl or palmitoyl-oleoylderivatives

IsoHistSuccDG 1 ^-Dipalmitoylglycerol-O-Histidinyl-Na-hemisuccinat, &

Distearoyl-, Dimyristoyl, Dioleoyl or palmitoyl- oleoylderivatives AC Acylcarnosine, Stearyl- & Palmitoylcarnosine

HCChol Nα-Histidinyl-Cholesterolcarbamate

Any dialkyl derivatives of the amphoteric lipids comprising diacyl groups listed above are also within the scope of the present invention.

Brief description of the drawings

Figure 1 shows IC 50 values of amphoteric liposomes encapsulating siRNA targeting Plk-1 derived from the in vitro transfection of HeIa cells as described in example 6. Shown are mixtures of MoChol/DMGS, CHIM/DMGS, CholC4N-Mo2/DMGS and CholC3N-Mo2/DMGS with a C/A ratio of 0.33 and increasing amounts of Choi as neutral lipid (0-60 mol%). The bars marked with an asterisk indicate that the IC 50 values are higher than the tested dose range (75 or 100 nM siRNA, respectively).

Figure 2 shows IC 50 values of amphoteric liposomes encapsulating siRNA targeting Plk-1 derived from the in vitro transfection of HeIa cells as described in example 6. Shown are mixtures of MoChol/DMGS, CHIM/DMGS, CholC4N-Mo2/DMGS and CholC3N-Mo2/DMGS with a C/A ratio of 0.5 and increasing amounts of Choi as neutral lipid(0-60 mol%). The bars marked with an asterisk indicate that the IC 50 values are higher than the tested dose range (75 or 100 nM siRNA, respectively).

Figure 3 shows IC 50 values of amphoteric liposomes encapsulating siRNA targeting Plk-1 derived from the in vitro transfection of HeIa cells as described in example 6.

Shown are mixtures of MoChol/DMGS, CHIM/DMGS, CholC4N-Mo2/DMGS and CholC3N-Mo2/DMGS with a C/A ratio of 0.33 and increasing amounts of POPC/Chol=0.5 (molar ratio) as neutral lipid(0-60 mol%). The bars marked with an asterisk indicate that the IC 50 values are higher than the tested dose range (75 nM siRNA).

Figure 4 shows IC 50 values of amphoteric liposomes encapsulating siRNA targeting Plk-1 derived from the in vitro transfection of HeIa cells as described in example 6. Shown are mixtures of MoChol/DMGS, CHIM/DMGS, CholC4N-Mo2/DMGS and CholC3N-Mo2/DMGS with a C/A ratio of 0.5 and increasing amounts of POPC/Chol=0.5 (molar ratio) as neutral lipid (0-60 mol%). The bars marked with an asterisk indicate that the IC 50 values are higher than the tested dose range (75 or 100 nM siRNA, respectively).

Figure 5 shows the % cell viability (normalized to mock treated cells) of HeIa cells transfected with different amphoteric liposomes encapsulating siRNA targeting Plk-1 (black bars) or non-targeting scrambled siRNA (grey bars). Shown are following amphoteric liposome formulations: MoChol/DMGS/Chol 17.5:52.5:30 (molar ratio), CHIM/DMGS/Chol 17.5:52.5:30 (molar ratio),CholC4N-Mo2/DMGS/Chol 17.5:52.5:30 (molar ratio) and CholC3N-Mo2/DMGS/Chol 17.5:52.5:30 (molar ratio).

Figure 6 shows the % cell viability (normalized to mock treated cells) of HeIa cells transfected with different amphoteric liposomes encapsulating siRNA targeting Plk-1 (black bars) or non-targeting scrambled siRNA (grey bars). Shown are following amphoteric liposome formulations: MoChol/DMGS/POPC/Chol 26.7:53.3:6.7:13.3 (molar ratio), CHIM/DMGS/POPC/Chol 26.7:53.3:6.7:13.3 (molar ratio), CholC4N- Mo2/DMGS/POPC/Chol 26.7:53.3:6.7:13.3 (molar ratio), CholC3N- Mo2/DMGS/POPC/Chol 26.7:53.3:6.7:13.3 (molar ratio).

Examples are given with the understanding of further detailing certain aspects of practising the current invention. The examples by no means limit the scope of this disclosure.

Example 1 :

Synthesis of [^-Morpholine^-yl-ethylcarbamoyOmethylJ-carbamic acid cholesteryl ester (D

Reaction scheme:

Step 1 : Synthesis of [(2-Morpholine-4-yl-ethylcarbamoyl)methyl]-carbamic acid tert- butyl ester

6 g N-tert.-butyloxycarbonyl-glycine and 12.65 g TBTU (2-(1 H-benzotriazole-1-yl)- 1 ,1 ,3,3-tetramethyluronium tetrafluoroborate) were suspended in 200 ml THF (tetrahydrofurane) and stirred. Then 4.46 g 4-(2-aminoethyl)morpholine und 10.39 g N-methylmorpholine were added. The reaction mixture was allowed to stir at room temperature overnight. The suspension was filtered and the solvent of the filtrate was removed by rotary evaporation. The crude product was purified by column chromatography at silica gel (eluent: ethyl acetate : methanol 1 :1 + 1% NH ₄ OH). The product, a colourless oil, was characterized by ¹ H-NMR.

Step 2: Synthesis of 2-Amino-N-(2-morpholine-4-yl-ethyl)-acetamide

Under vacuum 9.8 g [(2-Morpholine-4-yl-ethylcarbamoyl)methyl]-carbamic acid tert- butyl ester were dissolved in 280 ml dry dichloromethane into a round bottom flask.

The mixture was cooled with an ice bath and stirred for 50 min. Subsequently, 52.56 ml trifluoroacetic acid were added drop wise to the mixture. The ice bath was removed and the reaction was allowed to stir for 2 h. The solvent was removed by rotary evaporation and without further purification the product was used for step 3 of the synthesis.

Step 3: Synthesis of [(2-Morpholine-4-y!-ethy!carbamoy!)methy!]-carbarriic acid cholesteryl ester (1)

2.56 g 2-Amino-N-(2-morpholine-4-yl-ethyl)-acetamid and 100 ml chloroform were added under vakkum into a round bottom flask and the mixture was cooled with an ice bath. Then 8.65 g triethylamine were added drop wise to the mixture. Subsequently, 4.8 g cholesteryl chloroformiate, dissolved in 25 ml chloroform, were added drop wise. The reaction mixture was stirred for 1.5 days. Then the solvent was removed by rotary evaporation and the crude product purified by column chromatography at silica gel (eluents: CHCI ₃ + 1 ,5 % MeOH; CHCI ₃ + 2 % MeOH). The product was characterized by ¹ H-NMR and LC-MS.

Example 2:

Synthesis of β-^-Morpholine^-yl-ethylcarbamoylJ-ethylJ-carbamic acid cholesteryl ester (2)

Reaction scheme:

Step 1 : Synthesis of [2-(2-Morpholine-4-yl-ethylcarbamoyl)-ethyl]-carbamic acid tert- butyl ester

6 g N-tert.-Butyloxycarbonyl-beta-alanine and 11.71 g TBTU were suspended in 200 ml THF and stirred. Then 4.13 g 4-(2-aminoethyl)morpholine and 9.62 g N- methylmorpholine were added and the reation mixture was allowed to stir at room temperature over night. The suspension was filtered and the solvent of the filtrate was removed by rotary evaporation. The crude product was purified by column

chromatography at silica gel (eluent: ethyl acetate : methanol 1 :1 + 1% NH ₄ OH). The product, a colourless oil, was characterized by ¹ H-NMR.

Step 2: Synthesis of 3-Amino-N-(2-morpholine-4-yl-ethyl)-propionamid ditrifluoroacetate

6.6 g [2-(2-Morpholine-4-yl-ethylcarbamoyl)-ethyl]-carbamic acid tert.-butyl ester were added into a round bottom flask and under vacuum dissolved in 200 ml dry dichloromethane. The mixture was cooled and 33.74 ml thfluoroacetic acid were added drop wise to the mixture. The ice bath was removed and the reaction was allowed to stir for 5 h at room temperature. Then the solvent was removed by rotary evaporation and without further purification the product, a yellow oil, was used for step 3 of the synthesis.

Step 3: Synthesis of [2-(2-Morpholine-4-yl-ethylcarbamoyl)-ethyl]-carbamic acid cholesteryl ester (2)

5.8 g 3-amino-N-(2-morpholine-4-yl-ethyl)-propionamid ditrifluoroacetate were dissolved in 150 ml chloroform. Then 11.09 g triethylamine and 3.94 g cholesteryl chloroformiate were added. The reaction mixture was stirred at room temperature overnight. Then the solvent was removed by rotary evaporation and the crude product, a colourless oil, dissolved in 200 ml ethyl acetate and washed twice with water. The organic phase was dried over Na ₂ SO ₄ overnight and after filtration the solvent was removed by rotary evaporation. The product was dried by lyophilization and characterized by ¹ H-NMR and LC-MS.

Example 3:

Synthesis of [(S-Morpholine^-yl-propylcarbamoyO-methylJ-carbamic acid cholesteryl ester (3)

Reaction scheme:

3

Step 1 : Synthesis of [(S-Morpholine^-yl-propylcarbamoyO-methyll-carbamic acid tert.-butyl ester

6 g N-tert.-butyloxycarbonyl-glycine and 12.65 g TBTU were suspended in 200 ml THF and stirred. Then 4.94 g N-(3-aminopropyl)morpholine and 10.39 g N- methylmorpholine were added and the reaction mixture was allowed to stir at room temperature over night. The suspension was filtered and the solvent of the filtrate was removed by rotary evaporation. The crude product was purified by column chromatography at silica gel (eluent: ethyl acetate : methanol 1 :1 + 1% NH ₄ OH). The product, a colourless oil, was characterized by ¹ H-NMR.

Step 2: Synthesis of 2-Amino-N-(3-morpholine-4-yl-propyl)-acetamid bistrifluoroacetate

9.82 g [(S-Morpholine^-yl-propylcarbamoylJ-methylJ-carbamic acid tert.-butyl ester were added into a round bottom flask and under vacuum dissolved in 250 ml dry dichloromethane. The mixture was cooled with an ice bath and 50.2 ml trifluoroacetic acid were added. The ice bath was removed and the reaction was allowed to stir for 3.5 h at room temperature. Then the solvent was removed by rotary evaporation and without further purification the product, a yellow oil, was used for step 3 of the synthesis.

Step 3: Synthesis of [(3-Mθφholine-4-yl-propylcarbamoyl)-methyl]-carbamic acid cholesteryl ester (3)

5.8 g 2-amino-N-(3-morpholine-4-yl-propyl)-acetamid bistrifluoroacetate were dissolved in 150 ml chloroform. Then 13.3 g triethylamine and 3.94 g cholesteryl chloroformiate were added. The reaction mixture was stirred at room temperature overnight. Then the solvent was removed by rotary evaporation and the crude product, a colourless oil, dissolved in 200 ml ethyl acetate and washed twice with water. The organic phase was dried over Na ₂ SO ₄ and after filtration the solvent was removed by rotary evaporation. The product was dried by lyophilization and characterized by ¹ H-NMR and LC-MS.

Example 4:

Synthesis of [i-Methyl-Z-^-morpholine^-yl-ethylcarbamoyO-propylj-carbamic acid cholesteryl ester (4)

Reaction scheme:

Step 1 : Synthesis of 3-Amino-2-methyl-butyric acid

In a round bottom flask with a reflux condenser to 3.55 g sodium (in small pieces) 125 ml dry ethanol was added. When all sodium was dissolved and the solution cooled down to room temperature 10.73 g hydroxylamine ^* HCI in 8 ml H ₂ O were heated to 60 ⁰ C and then added to the reaction mixture. The reaction was cooled with an ice bath and then centrifuged. The supernatant was added to 7.73 g (E)-2- methyl-but-2-enoic acid and the yellow solution was refluxed at 100 ⁰ C overnight and then cooled down to room temperature. After the addition of a couple of seed crystals the mixture was incubated at -20 ⁰ C for 7 h. The formed crystals were filtered,

washed with 10 ml cold ethanol and then dried. The product was characterized by thin layer chromatography.

Step 2: Synthesis of S-tert.-Butoxycarbonylamino^-methyl-butyric acid

2.65 g 3-amino-2-methyl-butyric acid were added into a round bottom flask and cooled with an ice bath. Then 2.64 g sodium carbonate (anhydrous) dissolved in 25 ml H ₂ O were added slowly to the 3-amino-2-methyl-butyric acid. In addition, 30.9 g 1.4-dioxane and 4.94 g di-tert-butyl-dicarbonate were added to the mixture. The ice bath was removed and the mixture was allowed to stir at room temperature for 2 days. Then 1 ,4-dioxane was removed by rotary evaporation and subsequently the mixture was diluted with 20 ml H ₂ O. After an extraction with diethyl ether the aqueous phase was overlaid with ethyl acetate and acidified to pH 3 with a KHSO ₄ solution. The formed white precipitate was removed by filtration and after a separation of the phases the aqueous phase was extracted twice with ethyl acetate. Then the organic phases were combined and the solvent removed by rotary evaporation. The white residue was then combined with the precipitate above and dried. This product was characterized by ¹ H-NMR.

Step 3: Synthesis of [1-Methyl-2-(2-Morpholine-4-yl-ethylcarbamoyl)-propyl]-carba mic acid tert. butyl ester

Under vacuum 3.3.g 3-tert.-Butoxycarbonylamino-2-methyl-butyhc acid and 5.85 g TBTU were suspended in 100 ml THF and stirred. Then 2.18 g 4-(2- aminoethyl)morpholine und 4.61 g N-methylmorpholine were added and the reaction mixture was allowed to stir at room temperature over night. The suspension was filtered and the solvent of the filtrate was removed by rotary evaporation. The residue was purified twice by column chromatography at silica gel (eluents: ethyl acetate : methanol 1 :1 ; ethyl acetate : methanol 2.5:1). The crude product was dissolved in ethyl acetate and this organic phase was washed with a saturated Na ₂ CO ₃ -solution and with brine. After drying the organic phase over Na ₂ SO ₄ the solvent was removed by rotary evaporation and a white product remained.

Step 4: Synthesis of 3-Amino-2-methy!-N-(2-morpho!ine-4-y!-ethyl)-butyramide bistrifluoroacetate

Under vacuum 6.2 g [1-Methyl-2-(2-Morpholine-4-yl-ethylcarbamoyl)-propyl]- carbamic acid tert. butyl ester and 200 ml dry dichloromethane were added into a round bottom flask. Then the mixture was cooled with an ice bath and 29 ml trifluoroacetic acid were added drop wise. The ice bath was removed and the reaction was allowed to stir for 2 h at room temperature. Then the solvent was removed by rotary evaporation and the product, a yellow oil, characterized by ¹ H- NMR.

Step 5: Synthesis of [i-Methyl^^-Morpholine^-yl-ethylcarbamoylJ-propy^-carbamic acid cholesteryl ester (4)

Under vacuum 6.95 g 3-amino-2-methyl-N-(2-morpholine-4-yl-ethyl)-butyramide bistrifluoroacetate were dissolved in 90 ml chloroform and cooled with an ice bath. Then 15.3 g triethylamin und von 5.69 g cholesteryl chloroformiate, dissolved in 20 ml chloroform, were added slowly to the mixture. The ice bath was removed and the reaction mixture was allowed to stir at room temperature for 2.5 days. Then the solvent was removed by rotary evaporation and the crude product purified twice by column chromatography at silica gel (eluents: CHCI ₃ + 1 % methanol; CHCI ₃ + 2 % methanol). The product was characterized by ¹ H-NMR and LC-MS.

Example 5:

Stability of the inventive lipids

Preparation of lipid suspensions:

The lipids (see table 6) were dissolved in isopropanol to a final lipid concentration of 30 mM. The lipid solutions were then mixed with PBS resulting in a final lipid concentration of 1 mM and a final isopropanol amount of 3 %.

Stability test:

Stability of the lipids was tested under stress conditions, including acidic, alkaline and oxidative conditions at room temperature. In addition, one aliquot of the lipid suspensions was incubated at 60 ⁰ C for three days. The conditions of the stability test are summarized in table 5 below.

Table 5:

HPTLC: After the incubation of the samples under the above mentioned conditions samples were analyzed by HPTLC (High Performance Thin Layer Chromatography).

Results:

The results of the stability test are summarized in table 6 as % degradation of the appropriate lipid.

Table 6:

Compared to Mochol the inventive lipids show an improved stability, especially under alkaline conditions and under heat at physiological pH.

Example 6:

In vitro transfection of HeIa cells with amphoteric liposomes encapsulating siRNA targeting Plk-1 or non-targeting scrambled (scr) siRNA

Amphoteric liposomes comprising lipids according to the present invention were used for tranfection of HeIa cells.

Preparation of liposomes:

Liposomes were manufactured by an isopropanol-injection method. Lipids were dissolved in isopropanol (30 mM lipid concentration) and mixed. Liposomes were produced by adding siRNA solution in NaAc 2OmM, Sucrose 300 mM, pH 4.0 (pH adjusted with HAc) to the alcoholic lipid mix, resulting in a final alcohol concentration of 30% and a N/P ratio 5. The formed liposomal suspensions were shifted to pH 7.5 with twice the volume of Na2HPO4 136mM, NaCI 10OmM (pH 9), resulting in a final lipid concentration of 3 mM and a final isopropanol concentration of 10%.

N/P = the ratio cationic charges from the lipids to anionic charges from the siRNA during manufacturing.

Plk-1 siRNA as in Haupenthal et al., lnt J Cancer, 121 , 206-210 (2007).

Following liposomal formulations were prepared with Plk-1 siRNA or scrambled siRNA and tested for transfection efficiency on HeIa cells:

Table 7:

Transfection protocol:

HeLa cells were obtained from DSMZ (German Collection of Micro Organism and Cell Cultures) and maintained in DMEM. Media were purchased from Gibco- Invitrogen and supplemented with 10% FCS. The cells were plated at a density of 2.5*10 ⁴ cells/ml and cultivated in 100 μl medium at 37 ⁰ C under 5% CO ₂ . After 16 h the liposomes containing siRNA were diluted in the manufacturing buffer system and then 10 μl were added to the cells (110μl final Volume and 9.1 % FCS per well) (0,4 to 100 nM PIkI or scrambled siRNA). 10μl dilution buffer were also added to untreated cells and into wells without cells. In addition, as control, free siRNA was added to the cells (10 to 80 nM Plk-1 or scrambled siRNA). Cell culture dishes were incubated for 72 h hours at 37 ⁰ C under 5% CO ₂ . Transfection efficiency was analyzed using a cell proliferation/viability assay.

Cell proliferation/viability assay:

Cell proliferation/viability was determined by using the CellTiter-Blue Cell Viability assay (Promega, US). In brief, 72 hours after transfection, 100 μl Medium/CellTiter- Blue reagent (Pre-mix of 80ml Medium and 20μl CellTiter-Blue reagent) were added to the wells. Following an incubation at 37 ⁰ C for 2.5 hours, 80 μl of the medium were

transferred into the wells of a black microtiter plate (NUNC, Denmark). Fluorescence was recorded using a fluorescence plate reader (Ex. 550 nm/Em. 590 nm). On each plate the following controls were included: i) wells without cells but with medium (control for culture medium background fluorescence) and ii) wells with cells (untreated cells = mock-transfected cells). For calculation, the mean fluorescence value of the culture medium background was subtracted from all mean (triplicates) values of experimental wells (transfected and mock-transfected cells). The fluorescence values from each transfection were normalized to the mean fluorescence value from mock-transfected cells, which was set as being 100%.

Results:

Figs. 1-6 show the results of the transfection experiments. Compared to the lipids MoChol or CHIM the transfection efficiency can be improved by using the inventive novel sterol lipids (see IC 50 values shown in Figs. 1-4). Figs. 5 and 6 show the dose response curves from specific liposomal formulations of the four tested cationic sterols comprising Plk-1 siRNA or scrambled siRNA.