HK1119090B - Coated lipid complexes and their use - Google Patents

Description

Coated lipid complexes and their use

The present invention relates to cationic lipids, compositions containing cationic lipids and uses thereof, and to methods for delivering compounds into cells.

Both molecular biology and molecular medicine rely heavily on the introduction of biologically active compounds into cells. Such biologically active compounds typically comprise, among others, DNA, RNA, and peptides and proteins. The obstacle that has to be overcome is typically a lipid bilayer with a negatively charged outer surface. In the art, a number of techniques have been developed to penetrate cell membranes and thereby introduce the biologically active compounds. However, some of the methods envisaged for laboratory use cannot be used in the medical field and are more particularly not suitable for drug delivery. For example, electroporation and ballistic methods known in the art will only allow for local delivery of biologically active compounds, if at all. In addition to the lipid bilayer cell membrane, a transport system is also included. Therefore, efforts have been made to use this kind of transport system in order to deliver the biologically active compound across the cell membrane. However, due to the specificity or cross-reactivity of such transport systems, their use is not a generally applicable approach.

A more generally applicable method described in the art for transferring biologically active compounds into cells is the use of viral vectors. However, viral vectors can only be used to efficiently transfer genes into some cell types; they cannot be used to introduce chemically synthesized molecules into the cell.

An alternative approach is to use so-called liposomes (Bangham, J.Mol.biol.13, 238-. Liposomes are vesicles that are created when amphiphilic lipids associate in water. Liposomes typically comprise concentrically arranged phospholipid bilayers. Depending on the number of layers, liposomes can be classified as small single lamellar vesicles, multi lamellar vesicles and large single lamellar vesicles. Liposomes have proven to be effective delivery agents because they can incorporate hydrophilic compounds into the aqueous middle layer, while incorporating hydrophobic compounds into the lipid layer. It is well known in the art that the composition of a lipid formulation and its method of preparation has an effect on the structure and size of the resulting lipid aggregates and thus on the liposomes. Liposomes are also known to incorporate cationic lipids.

In addition to being a component of liposomes, cationic lipids have attracted considerable attention because they can thus be used for cellular delivery of biopolymers. With cationic lipids, essentially any anionic compound can be encapsulated in a quantitative manner due to electrostatic interactions. In addition, it is believed that the interaction of cationic lipids with negatively charged cell membranes triggers cell membrane transport. It has been found that the use of liposomal formulations containing cationic lipids or the use of cationic lipids as such in conjunction with biologically active compounds requires heuristics, as each formulation is of limited utility, as it can typically deliver plasmids to some but not all cell types, usually in the absence of serum.

The ratio of the charge and/or mass of the lipids and biologically active compounds to be transported through them has proven to be a key factor in the delivery of different types of said biologically active compounds. For example, lipid formulations suitable for plasmid delivery comprising 5,000 to 10,000 bases in size have been shown to be generally not effective for delivery of oligonucleotides such as synthetic ribozymes or antisense molecules typically comprising about 10 to about 50 bases. In addition, it has recently been shown that the optimal delivery conditions for antisense oligonucleotides and ribozymes are different even in the same cell type.

U.S. Pat. No. 6,395,713 discloses cationic lipids based on compositions typically consisting of lipophilic groups, linkers and head groups, and the use of such compositions for the transfer of biologically active compounds into cells.

The problem underlying the present invention is to provide a method for introducing biologically active compounds into cells, preferably animal cells. A further problem underlying the present invention is to provide delivery agents for nucleic acids, in particular small nucleic acids such as siRNA, siNA and RNAi or aptamers and spiegelmers.

A further problem underlying the present invention is to provide a delivery agent with good transfection and delivery characteristics while providing a long circulation time in vivo.

These problems are solved by the subject matter of the independent claims incorporated herein. Preferred embodiments can be taken from the dependent claims appended hereto.

In a first aspect, the problem underlying the present invention is also solved by a lipid composition comprising

At least one first lipid component selected from the group consisting of,

at least one first helper lipid, and

a shielding compound that is removable from the lipid composition under in vivo conditions.

In one embodiment of the first aspect, the shielding compound is selected from the group comprising PEG, HEG, polyhydroxyethyl starch (polyHES) and polypropylene.

In a preferred embodiment of the first aspect, the shielding compound is PEG2000 or PEG 5000.

In one embodiment of the first aspect, the composition comprises a further component and/or a second helper lipid.

In one embodiment of the first aspect, the lipid composition comprises a nucleic acid, wherein such a nucleic acid is preferably the further component.

In a preferred embodiment of the first aspect, the nucleic acid is selected from the group comprising RNAi, siRNA, siNA, antisense nucleic acids, ribozymes, aptamers and spiegelmers.

In one embodiment of the first aspect, the shielding compound is a conjugate of PEG and ceramide.

In a preferred embodiment of the first aspect, the ceramide comprises at least one short carbon chain substituent of 6 to 10 carbon atoms, preferably 8 carbon atoms.

In one embodiment of the first aspect, the ceramide is a first helper lipid.

In an alternative embodiment of the first aspect, the ceramide is a second helper lipid.

In one embodiment of the first aspect, the shielding compound is bound to the nucleic acid.

In a preferred embodiment of the first aspect, the shielding compound is bound to the nucleic acid via a linker moiety, preferably covalently bound to the nucleic acid via a linker moiety.

In a more preferred embodiment of the first aspect, said linker moiety is selected from the group comprising ssRNA, ssDNA, dsRNA, dsDNA, peptides, S-linkers and pH sensitive linkers.

In a preferred embodiment of the first aspect, the nucleic acid is selected from the group comprising RNAi, siRNA and siNA, and the linker is bound to the 3' end of the sense strand.

In a preferred embodiment of the first aspect, the shielding compound comprises a pH-sensitive linker or a pH-sensitive moiety.

In a preferred embodiment of the first aspect, the linker or moiety is an anionic linker or anionic moiety.

In a more preferred embodiment of the first aspect, the linker or anionic moiety of the anion is weakly anionic or neutral in an acidic environment, wherein preferably such acidic environment is an endosome.

In one embodiment of the first aspect, the pH-sensitive linker or pH-sensitive moiety is selected from the group comprising oligo (glutamic acid), oligo phenolate and diethylenetriaminepentaacetic acid.

In one embodiment of the first aspect, the first lipid component is a compound according to formula (I),

wherein R is₁And R₂Each and independently selected from the group comprising alkyl groups;

n is any integer between 1 and 4;

R₃is an acyl group selected from the group comprising lysyl, ornithyl, 2, 4-diaminobutyryl, histidyl and an acyl moiety according to formula (II),

wherein m is any integer of 1 to 3, wherein NH₃ ⁺Optionally is absent, and

Y^-pharmaceutically acceptable yinIons.

In a preferred embodiment of the first aspect, R₁And R₂Each and independently selected from the group comprising lauryl, myristyl, palmityl and oleyl.

In a more preferred embodiment of the first aspect, R₁Is lauryl and R₂Is myristyl; or

R₁Is palmityl and R₂Is oleyl.

In a more preferred embodiment of the first aspect, m is 1 or 2.

In a more preferred embodiment of the first aspect, the compound is a cationic lipid, preferably with an anion Y^-And (4) associating.

In a more preferred embodiment of the first aspect, Y^-Selected from the group comprising halides, acetates and trifluoroacetates.

In a more preferred embodiment of the first aspect, the compound is selected from the group comprising:

-beta-arginyl-2, 3-aminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride

-beta-arginyl-2, 3-aminopropionic acid-N-lauryl-N-myristyl-amide trihydrochloride

And

-epsilon-arginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride

In a more preferred embodiment of the first aspect, the composition comprises a carrier.

The problem underlying the present invention is also solved in the second aspect by a pharmaceutical composition comprising a composition according to the first aspect and a pharmaceutically active compound and preferably a pharmaceutically acceptable carrier.

In one embodiment of the second aspect, the pharmaceutically active compound and/or the further component is selected from the group comprising peptides, proteins, oligonucleotides, polynucleotides and nucleic acids.

In a preferred embodiment of the second aspect, the protein is an antibody, preferably a monoclonal antibody.

In an alternative preferred embodiment, the nucleic acid is selected from the group comprising DNA, RNA, PNA and LNA.

In a preferred embodiment of the second aspect, the nucleic acid is a functional nucleic acid, wherein preferably a functional nucleic acid selected from the group comprising RNAi, siRNA, siNA, antisense nucleic acids, ribozymes, aptamers and spiegelmers.

In an embodiment of the first and second aspect, the first helper lipid and/or the second helper lipid is selected from the group comprising phospholipids and steroids, preferably with the proviso that the first and/or second helper lipid is different from a ceramide.

In a preferred embodiment of the first and second aspect, the first and/or second helper lipid or helper lipid component is selected from the group comprising 1, 2-diphytanoyl-sn-trioxy-3-phosphoethanolamine and 1, 2-dioleyl-sn-trioxy-3-phosphoethanolamine.

In a more preferred embodiment of the first and second aspect, the content of the helper lipid component is from about 20 mole% to about 80 mole% of the total lipid content of the composition.

In a still more preferred embodiment of the first and second aspect, the content of the helper lipid component is from about 35 mol% to about 65 mol%.

In one embodiment of the first and second aspect, the lipid is β -arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride and the helper lipid is 1, 2-diphytanoyl-sn-trioxy-3-diphosphoxyphospholethanolamine.

In a preferred embodiment of the first and second aspect, the lipid is 50 mol% and the helper lipid is 50 mol% of the total lipid content of the composition.

In one embodiment of the first and second aspects, the composition further comprises a second helper lipid.

In one embodiment of the first and second aspect, the first and/or second helper lipid comprises a group selected from the group comprising a PEG moiety, a HEG moiety, a polyhydroxyethyl starch (polyHES) moiety and a polypropylene moiety, wherein such moiety preferably provides a molecular weight of about 500 to 10000Da, more preferably about 2000 to 5000 Da.

In a preferred embodiment of the first and second aspect, the helper lipid comprising a PEG moiety is selected from the group comprising 1, 2-distearoyl-sn-propanetrioxy-3-diphosphoethanolamine and 1, 2-dialkyl-sn-propanetrioxy-3-diphosphoethanolamine.

In a more preferred embodiment of the first and second aspect, the PEG moiety of the helper lipid has a molecular weight of 2,000 to 5,000Da, preferably a molecular weight of 2,000 Da.

In a still more preferred embodiment of the first and second aspect, the composition comprises as the lipid component β -arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride, 1, 2-diphytanoyl-sn-trioxy-3-phosphoethanolamine as a first helper lipid and 1, 2-distearoyl-sn-trioxy-3-phosphoethanolamine-PEG 2000 as a second helper lipid.

In one embodiment of the first and second aspects, the second helper lipid is present in an amount of about 0.05 to 4.9 mol%, preferably about 1 to 2 mol%.

In one embodiment of the first and second aspects, the composition contains about 1 to 10 mole%, more preferably 1 to 7.5 mole% and most preferably 1 to 5 mole% of the conjugate of PEG and ceramide.

In a preferred embodiment of the first and second aspects, the ceramide is C8m and the PEG is PEG2000, and wherein the content of the conjugate of PEG and ceramide is about 1 to 7.5 mole%.

In an alternative preferred embodiment of the first and second aspect, the ceramide is C8m and the PEG is PEG5000, and wherein the content of the conjugate of PEG and ceramide is about 1 to 5 mole%.

In one embodiment of the first and second aspects, the first lipid component is present in an amount of about 42.5 to 50 mole%, and the first helper lipid is present in an amount of about 42.5 to 50 mole%, wherein the total amount of the first lipid component, the first helper lipid, and the conjugate of PEG and ceramide is 100 mole%.

In one embodiment of the first and second aspect, the functional nucleic acid is a double-stranded ribonucleic acid, wherein the composition further comprises a nucleic acid, preferably a functional nucleic acid, more preferably a double-stranded ribonucleic acid, and most preferably a nucleic acid selected from the group comprising RNAi, siRNA, siNA, antisense nucleic acids and ribozymes, wherein preferably the molar quantification of RNAi for cationic lipids is about 0 to 0.075, preferably about 0.02 to 0.05 and even more preferably 0.037.

In one embodiment of the first and second aspect, the first lipid component and/or at least one of the helper lipids and/or the shielding compound is present as a dispersion in an aqueous medium.

In an alternative embodiment of the first and second aspect, the first lipid component and/or at least one of the helper lipids and/or the shielding compound is present as a solution in a water-miscible solvent, wherein preferably the solvent is selected from the group comprising ethanol and tert-butanol.

In a third aspect, the problem underlying the present invention is also solved by the use of a composition according to the first or second aspect for the manufacture of a medicament.

In a fourth aspect, the problem underlying the present invention is also solved by using a composition according to the first or second aspect as transfer agent.

In one embodiment of the fourth aspect, the transfer agent transfers the pharmaceutically active component and/or the further component into a cell, preferably a mammalian cell and more preferably a human cell.

In a fifth aspect, the problem underlying the present invention is also solved by a method for transferring a pharmaceutically active compound and/or a further component into a cell or across a membrane, preferably a cell membrane, said method comprising the steps of:

-providing a cell or membrane;

-providing a composition, a pharmaceutically active compound and/or further components according to the first or second aspect; and

-contacting the cell or the membrane with a composition according to the first or second aspect.

In one embodiment of the fifth aspect, the method comprises the further steps of:

-detecting the pharmaceutically active compound and/or the further component on the cells and/or the membrane (beyond).

The inventors of the present invention have surprisingly found that a very efficient delivery of nucleic acids, in particular small nucleic acids such as RNAi, siRNA and siNA, by in vivo administration can be achieved by using a lipid composition comprising at least one first lipid component, at least one first helper lipid and a shielding compound. The shielding compounds provide longer circulation times in vivo and thus facilitate better biodistribution of the nucleic acids comprising the lipid composition. By this mechanism, it is possible to locate a site in the human or animal body which is relatively far from the site of administration of such a lipid composition, since the lipid composition is not immediately absorbed by the tissue surrounding the injection site, which is usually the endothelial lining of the vasculature when administered intravenously. A shielding compound as used herein is preferably a compound that avoids direct interaction of the lipid composition with serum compounds or other body fluids or compounds of the cytoplasmic membrane, preferably the cytoplasmic membrane of the endothelial lining of the vasculature into which the lipid composition is preferably administered. The term shielding also means that elements of the immune system do not immediately interact with the lipid composition, again increasing its circulation time in the living organism. Provided that the shielding compound acts as an anti-opsinization compound. Without wishing to be bound by any mechanism or theory, it appears that the shielding compound forms a covering or coating that reduces the surface area of the lipid composition for interaction with its environment, which would otherwise at the same time lead to fusion of the lipid composition with other lipids or binding of factors of the human and animal body, respectively, which is too early for such interaction, although it has to be acknowledged that at a later stage, i.e. after an extended time on administration of the lipid composition, such interaction is usually preferred to a certain extent in order to provide the delivery. It appears that another mechanism on which the observed efficacy of the shielding compound is based is the shielding of the overall charge of the lipid composition and in particular of the lipid composition containing nucleic acids, preferably functional nucleic acids as defined herein and more preferably siRNA, siRNA and RNAi. Furthermore, the interaction of the lipid composition appears to be influenced, wherein the effect arises from the shielding of the charge rather than the shielding of the lipid component. In connection with the present disclosure, it is recognized that a composition may comprise only one lipid.

The shielding compound is preferably a biologically inert compound. More preferably, the shielding compound does not carry any charge on its surface or on molecules like this. Particularly preferred shielding compounds are thus polyethylene glycols, hydroxyethyl glucosyl polymers, polyhydroxyethyl starch (polyHES) and polypropylene, wherein any of said compounds preferably has a molecular weight of about 500 to 10000Da, more preferably about 2000 to 5000 Da.

An important feature of the present invention is that the shielding compound can be removed from the lipid composition under in vivo conditions. Such removal exposes another component of the lipid composition, or a portion thereof, to the environment, such as animals and humans, and ultimately allows for the release and delivery of compounds, such as nucleic acids, contained in the lipid composition, respectively. The term in vivo conditions preferably refers to those conditions present in the animal or human body, preferably in any bodily fluid such as blood, interstitial and intracellular fluids, and/or present in endosomes and/or lysosomes. Depending on the design of the shielding compound, the removal can be influenced in a number of ways, as will be described in more detail below.

The additional effect due to the loss of the shielding agent, especially if the shielding agent is PEG, is due to the fact that providing a lipid dosage form with PEG, also referred to as adding polyethylene glycol, often results in an impaired functional delivery of the nucleic acid molecule to be delivered into the cytoplasm through such a lipid dosage form. The substantial presence of PEG in the lipid formulation impairs the uptake of endosomes of liposomes typically formed from the lipid formulation or interferes with the necessary endosomal detachment of nucleic acids contained in or associated with the lipid composition.

In one embodiment, the shielding compound is selected from the group comprising PEG, HEG, polyhydroxyethyl starch (polyHES) and polypropylene, which are bound, preferably covalently bound, to ceramides. The ceramide interacts with lipid compounds and, if present, helper lipids. As long as the ceramide can be the first helper lipid or the second helper lipid. Preferably, the ceramide conjugated to PEG is different from the first helper lipid. The ceramide is embedded into the lipid fraction of the lipid composition, which is preferably formed by the first lipid component and the first helper lipid and will be released at a certain rate due to the relatively short hydrocarbon chains. When the ceramide is thus released from the lipid composition, this is a shielding agent. Thus, in this embodiment, the in vitro conditions allow for the splitting of the lipid dosage form and thus provide for an extended in vivo lifetime or circulation time of the lipid composition.

In further embodiments, the shielding agent is bound to a nucleic acid contained in or associated with the lipid composition. Preferably, the shielding agent is covalently bound to the nucleic acid, most preferably via a linker. In a more preferred embodiment, the linker is designed such that it decomposes under physiological conditions, i.e. conditions that prevail in an animal or human organism. In a preferred embodiment, only a certain percentage of the nucleic acids actually possess such a polymer via the linker. Without wishing to be bound by any theory, it seems sufficient that only a certain number of nucleic acid molecules forming part of the lipid composition have to have such a large fraction in order to provide a shielding effect for the lipid composition. Preferably, the portion of the nucleic acid molecule is in the range of 0 to 20%, more preferably 3 to 10%, and even more preferably 6 to 10%. Preferred amounts of nucleic acid with a shielding agent (also referred to herein as a shielding compound) are about 0 to 3%, 3 to 6%, 6 to 10% and 10 to 20%, where% is mole% as used in this paragraph and throughout this application if not indicated to the contrary.

Such linkers may be single stranded RNA linkers, more preferably comprising 1 to 20 nucleotides, which will be cleaved by the RNA endonuclease activity present under or under in vivo conditions. In a further embodiment, the linker is formed from a single-stranded DNA linker that is also cleaved by a DNA endonuclease present in or under in vivo conditions. In further embodiments, the linker may be formed from double stranded RNA or double stranded DNA. The stability of the linker composed of nucleic acids is typically as follows: ssRNA < dsRNA < ssDNA < dsDNA. This stability diversity facilitates the specific design of the residence time of the thus modified nucleic acid and the lipid composition, respectively, including such linker.

In a further embodiment, the linker may be formed from an oligopeptide, polypeptide or protein, which is cleaved by a protease present in or under in vivo conditions. Still further embodiments provide linkers comprising an S-S-linkage that is sensitive to redox conditions.

In an even further embodiment, the linker is a pH sensitive linker. The concept of pH sensitive linkers resides in the observation of internalization of lipid compositions, which in principle can be lipoplexes (lipoplexs) or liposomes, which are any shielding agents that are clearly advantageous when protecting said lipid compositions, after which internalization can impose restrictions on further use of said compounds, thereby transferring into said cells. As used herein, the shielding agent is bound to a pH sensitive linker that is or is included in the anionic portion of the preferred embodiment. The charge of this anionic moiety is altered in an acidic environment such as an endosome. Because of this, the interaction of the pH-sensitive linker with the cationic lipid contained in the lipid dosage form based on electrostatics is also altered, usually reduced, which results in a more or less gradual release of the pH-sensitive linker from the cationic lipid containing lipid dosage form. Thus, the liposomes become devoid of shielding agents and can thereby exert an effect in the cell. This class of linkers comprises both linkers as are known in the art as such and new linkers of this class, i.e. having the characteristics of this class. Preferably, such a linker is any linker having charge characteristics that allow the linker to react as described above. Representative of such pH sensitive linkers include, but are not limited to, oligo (glutamic acid), oligo phenolate, and diethylenetriaminepentaacetic acid. Such linkers are coupled to the shielding agent, and such linkers are incorporated into the shielding agent and then are generally referred to as part of the shielding agent, as is known to those skilled in the art.

The compound used as the first lipid component in the lipid composition according to the invention, which is also referred to herein as a compound according to the invention, can be seen as comprising a lipophilic group formed by the moiety R1-N-R2, consisting of c (o) -CH (NH) as depicted in fig. 1³⁺)(CH₂)_nthe-NH moiety forms a linker group and the head group formed by the R3 moiety. The inventors of the present invention have surprisingly found that this class of compounds, which exhibit a positive charge at the linker group, is particularly suitable for transferring biologically active compounds across cell membranes and preferably into cells, more preferably animal cells. Also, the inventors of the present invention have surprisingly found that transfer mediated by a compound according to the present invention will be particularly effective if the biologically active compound is a nucleic acid, more preferably siRNA and siNA.

As preferably used herein, the term alkyl refers to a saturated aliphatic group containing 8 to 20 carbon atoms, preferably 12 to 18 carbon atoms, or a mono-or polyunsaturated aliphatic hydrocarbon group containing 8 to 30 carbon atoms, containing at least one double and triple bond, respectively. Thus, in a preferred embodiment, the term alkyl also encompasses alkenyl and alkynyl groups. Alkyl refers to both branched and unbranched, i.e., nonlinear or linear, alkyl groups. Preferred straight chain alkyl groups contain 8 to 30 carbon atoms. More preferred straight chain alkyl groups contain 12 to 18 carbon atoms. Preferred branched alkyl groups contain 8 to 30 carbon atoms, wherein the number of 8 to 30 carbon atoms refers to the number of carbon atoms forming the backbone of such branched alkyl groups. The backbone of the branched alkyl group contains at least one alkyl group as a branch off the backbone, the alkyl group being as defined herein, more preferably an alkyl group comprising a short chain alkyl group, more preferably an alkyl group comprising 1 to 6, more preferably 1 to 3 and most preferably 1C atom. More preferred are alkyl groups having from 12 to 18 carbon atoms in the backbone, said straight chain alkyl groups being as defined above. Particularly preferred alkyl groups are phytalkyl groups.

In another embodiment, the alkyl group is an unsaturated branched or unbranched alkyl group as defined above. More preferably, such unsaturated aliphatic hydrocarbon groups contain 1, 2 or 3 or 4 double bonds, with groups having one double bond being particularly preferred. Most preferred is oleyl, which is C18:1 Δ 9, i.e. an aliphatic hydrocarbon group having 18C atoms, where there is one double bond in cis configuration at position 9, rather than a single bond connecting C atom No. 9 to C atom No. 10.

As used herein, n is any integer between 1 and 4, which means that n can be 1, 2, 3, and 4. As used herein, m is any integer between 1 and 3, which means that m can be 1, 2, and 3.

It will be appreciated that the compounds according to the invention are preferably cationic lipids. More preferably, any NH or NH2 group present in the compounds according to the invention is present in protonated form. Typically, any positive charge of the compounds according to the invention is offset by the presence of an anion. Such anions may be monovalent or polyvalent anions. Preferred anions are halides, acetates and trifluoroacetates. The halides as used herein are preferably fluorides, chlorides, iodides and bromides. Most preferred is chloride. On association of the cationic lipid and the biologically active compound to be transferred into the cell, the halide anion is replaced by a biologically active compound which preferably exhibits one or several negative charges, although it will be understood that the total charge of the biologically active compound is not necessarily negative.

It is to be understood that any compound according to formula (I) comprises at least two asymmetric C atoms. It is within the scope of the present invention that any possible enantiomer of such compounds is disclosed herein, i.e., in particular R-R; S-S; R-S and S-R enantiomers.

The compounds according to the invention may form a composition or part of a composition, wherein such composition comprises a carrier. In such compositions, also referred to herein as lipid compositions, the compounds according to the invention are also referred to as lipid components. Such a carrier is preferably a liquid carrier. Preferred liquid carriers are aqueous and nonaqueous carriers. Preferred aqueous carriers are water, water buffer systems, more preferably buffer systems having physiological buffer strength and physiological salt concentration. Preferred nonaqueous carriers are solvents, preferably organic solvents such as ethanol and t-butanol. Without wishing to be bound by any theory, in principle any water-miscible organic solvent may be used. It will be appreciated that the composition, more particularly the lipid composition, may thus be present as or form liposomes.

The composition according to the invention may comprise one or more helper lipids, also referred to herein as a helper lipid component. The co-lipid component is preferably selected from the group comprising phospholipids and steroids. The phospholipids are preferably diesters and monoesters of phosphoric acid. Preferred components of phospholipids are phosphoglycerides and sphingolipids. Steroids, as used herein, are naturally occurring and synthetic compounds based on partially hydrogenated cyclopenta [ a ] phenanthrene. Preferably, the steroid contains 21 to 30C atoms. A particularly preferred steroid is cholesterol.

Particularly preferred helper lipids are 1, 2-diphytanoyl-sn-propanetrioxy-3-diphosphoethanolamine (DPhyPE) and 1, 2-dioleoyl-sn-propanetrioxy-3-Diphosphoethanolamine (DOPE).

Particularly preferred compositions according to the invention comprise any of beta-arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride [ #6], beta-arginyl-2, 3-diaminopropionic acid-N-lauryl-N-myristyl-amide trihydrochloride [ #11], or epsilon-arginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride [ #15] in combination with DPhyPE in an amount of about 90 to 20 mol%, preferably 80 mol%, 65 mol%, 50 mol% and 35 mol%, wherein the term mol% refers to the percentage of the total lipid content of the composition, i.e. the lipid content of the composition comprising the cationic lipids according to the invention and any additional lipids, the additional lipids include, but are not limited to, any helper lipid.

It is within the scope of the present invention that the composition according to the invention preferably comprises a compound according to the invention and/or one or several helper lipids as disclosed herein, wherein either the compound according to the invention, i.e. the cationic lipid, and/or the helper lipid component is present as a dispersion in an aqueous medium. Alternatively, the compound according to the invention, i.e. the cationic lipid, and/or the helper lipid component, is present as a solution in a water-miscible solvent. As an aqueous medium, it is preferred to use any aqueous carrier as disclosed herein. Preferred water-miscible solvents are any solvents which form a homogeneous phase with water in any proportion. Preferred solvents are ethanol and tert-butanol. It will be appreciated that the composition, more particularly the lipid composition, may thus be present as or form liposomes.

It is to be understood that in its various embodiments, the composition according to the present invention is and may thus also be used as a pharmaceutical composition. In the latter case, the pharmaceutical composition comprises a pharmaceutically active compound and optionally a pharmaceutically acceptable carrier. Preferably, such a pharmaceutically acceptable carrier may be selected from the group of carriers as defined herein in connection with the composition according to the invention. It will be understood by those skilled in the art that any composition as described herein, in principle, also serves as a pharmaceutical composition, provided that its components and any combination thereof are pharmaceutically acceptable. The pharmaceutical composition comprises a pharmaceutically active compound. Such pharmaceutically active compound may be the same as the further component of the composition according to the invention, which is preferably any biologically active compound, more preferably any biologically active compound as disclosed herein. The further component, the pharmaceutically active compound and/or the biologically active compound is preferably selected from the group comprising peptides, proteins, oligonucleotides, polynucleotides and nucleic acids.

Preferably, any such biologically active compound is a negatively charged molecule. The term negatively charged molecule is meant to include molecules having at least one negatively charged group that can form an ion pair with a positively charged group of a cationic lipid according to the present invention, although the inventors do not wish to be bound by any theory. In principle, the positive charge at the linker moiety may also have some influence on the structure of such lipids or any complex formed between the cationic lipid and the negatively charged molecule, i.e. the biologically active compound. In addition to that, the additional positive charge introduced into the lipids according to the present invention should help to increase the toxicity of the lipid compared to the cationic lipids disclosed in U.S. Pat. No. 6,395,713, as described by Xu Y, Szoka FC jr; biochemistry; 1996May 07, 35 (18): 5616-23. Contrary to what the skilled person would expect from the prior art documents, the compounds according to the invention are particularly suitable for the various purposes disclosed herein and in particular without any increased toxicity.

A peptide as preferably used herein is any polymer consisting of at least two amino acids covalently linked to each other, preferably by peptide bonds. More preferably, the peptide consists of two to ten amino acids. A particularly preferred embodiment of the peptide is an oligopeptide, which more preferably comprises from about 10 to about 100 amino acids. Proteins which are preferably used here are polymers composed of a plurality of amino acids which are covalently linked to one another. Preferably, such proteins comprise about at least 100 amino acids or amino acid residues.

Preferred proteins that can be used for linking to the cationic lipids and compositions according to the invention are any antibodies, preferably any monoclonal antibodies.

Particularly preferred biologically active compounds, i.e. pharmaceutically active compounds and such further components for linking to the composition according to the invention, are nucleic acids. Such nucleic acids may be DNA, RNA, PNA or any mixture thereof. More preferably, the nucleic acid is a functional nucleic acid. Functional nucleic acids as preferably used herein are nucleic acids which are not nucleic acid encoding for peptides and proteins, respectively. Preferred functional nucleic acids are all siRNA, siNA, RNAi, antisense nucleic acids, ribozymes, aptamers, and spiegelmers known in the art.

SiRNA is small interfering RNA, for example, as described in International patent application PCT/EP 03/08666. These molecules generally consist of a double-stranded RNA structure comprising between 15 and 25, preferably 18 to 23, nucleotide pairs that are base-paired with each other, i.e., substantially complementary to each other, typically mediated by Watson-Crick base-pairing. One strand of the double stranded RNA molecule is substantially complementary to a target nucleic acid, preferably mRNA, and the second strand of the double stranded RNA molecule is substantially identical to the extension of the target nucleic acid. The siRNA molecules may be flanked on each side and on each extended side, respectively, by a number of additional oligonucleotides, which, however, do not necessarily have to be base-paired with each other.

RNAi has essentially the same design as siRNA, however, the molecule is significantly longer than siRNA. RNAi molecules typically comprise 50 or more nucleotides and base pairs, respectively.

Another class of functional nucleic acids based on the same mode of action being active as siRNA and RNAi is siNA. siNA is described, for example, in the international patent application PCT/EP 03/074654. More specifically, the siNA corresponds to siRNA, whereby the siNA molecule does not comprise any nucleotides.

Antisense nucleic acids, as preferably used herein, are oligonucleotides that hybridize to a target RNA, preferably mRNA, based on base complementarity, thereby activating RNaseH. RNaseH is activated by phosphodiester and phosphorothioate-conjugated DNA. However, phosphodiester-linked DNA is rapidly degraded by cellular nucleases, with the exception of phosphorothioate-linked DNA. Antisense polynucleotides are thus only effective as DNA-RNA hybrid complexes. Preferred lengths of antisense nucleic acids range from 16 to 23 nucleotides. Examples of antisense oligonucleotides of this kind are described, among others, in U.S. Pat. No. 5,849,902 and U.S. Pat. No. 5,989,912.

Other groups of functional nucleic acids are ribozymes as catalytically active nucleic acids, preferably consisting of an RNA essentially comprising two parts. The first part shows catalytic activity, while the second part is responsible for specific interactions with the target nucleic acid. In the interaction of the target nucleic acid and the ribozyme moiety, typically by hybridization and the substantially complementary extension of the bases of the two hybridized strands in Watson-Crick base pairs, the catalytically active moiety may become active, meaning that it cleaves the target nucleic acid intramolecularly or intermolecularly in the case where the catalytic activity of the ribozyme is phosphodiester activity. Ribozymes, the use and design principles of which are known to those skilled in the art and are described, for example, in Doherty and Doudna (Annu. Ref. Biophys. Biomols Truct. 2000; 30: 457-75).

Still another group of functional nucleic acids are aptamers. Aptamers are D-nucleic acids, which are single-stranded or double-stranded, that specifically interact with a target molecule. For example, the manufacture or selection of aptamers is described in european patent EP 0533838. In contrast to RNAi, siRNA, siNA, antisense nucleotides and ribozymes, aptamers do not degrade any target mRNA, but specifically interact with the secondary and tertiary structures of target compounds such as proteins. Upon interaction with a target, the target typically exhibits a change in its biological activity. The length of the aptamer is generally in the range of as little as 15 to as much as 80 nucleotides, and preferably in the range of about 20 to about 50 nucleotides.

Another group of functional nucleic acids are spiegelmers, for example, as described in International patent application WO 98/08856. spiegelmers are aptamer-like molecules. In contrast to aptamers, however, spiegelmers either consist entirely or mostly of L-nucleotides rather than D-nucleotides. In addition, especially with regard to the possible lengths of spiegelmers, the same applies to spiegelmers, as outlined with regard to the aptamers.

As previously mentioned, the inventors of the present invention have surprisingly found that the compounds according to the present invention, and the respective compositions comprising such compounds, are particularly effective in transferring RNAi, and more specifically siRNA and siNA, into cells. It should be noted that, although not wishing to be bound by any theory, due to the specific molar percentage of helper lipids comprised in the lipid composition according to the invention, which helper lipids may be PEG-free helper lipids or in particular PEG-containing helper lipids, unexpected effects may be achieved, more particularly if the content of any such kind of helper lipid is comprised within the concentration ranges specified herein. In connection with this, it is particularly noteworthy that any delivery or transfection with such PEG-derived helper lipids containing compositions is particularly effective in delivering nucleic acids, preferably RNAi molecules, most preferably siRNA, siNA, antisense nucleotides and ribozymes, if the composition according to the invention contains a helper lipid comprising a PEG moiety.

The reason for this is that the inventors of the present invention have surprisingly found that liposomes containing more than about 4% of PEG-containing helper lipids are not active, whereas liposomes containing less than 4% (preferably less than 3% but more than 0%) mediate functional delivery. Basically, the inventors of the present invention found that the specific amount of PEG in the lipid composition according to the present invention is suitable to provide efficient transfection and delivery, respectively.

In a further aspect, the inventors of the present invention have surprisingly found that the lipid composition according to the present invention preferably as a lipid complex or liposome, preferably exhibits an overall cationic charge and thus an excess of at least one positive charge. More preferably, the lipid composition exhibits a negative: the positive charge ratio is from about 1: 1.3 to 1: 5. Thus, the present invention thus relates to further aspects of any lipid composition comprising at least one cationic lipid and one nucleic acid, preferably RNAi, siRNA or siNA or any functional nucleic acid as defined herein, having a negative: the positive charge ratio is about 1: 1.3 to 1: 5. The cationic lipid is preferably any of the cationic lipids described herein. In a preferred embodiment, the lipid composition comprises any of the helper lipids or helper lipid combinations described herein.

The inventors of the present invention have also found that, in particular, the molar ratio of siRNA and cationic lipid may be crucial for the successful application of the lipid composition according to the present invention, in particular in view of what is said above with respect to the cationic overall charge of the nucleic acid comprising the lipid dosage form. Without wishing to be bound by any theory, it appears that, in particular as disclosed herein, 1 mole of cationic lipid can provide up to three positive charges per molecule, whereas the nucleic acid, and in particular the siRNA molecule as disclosed herein, provides up to 40 negative charges per molecule. To achieve the overall positive charge of the siRNA containing the lipid formulation according to the present invention, the molar ratio may range from 0 to a maximum of 0.075. Preferred molar ratio ranges are from about 0.02 to 0.05, and even more preferred is a molar ratio range of about 0.037.

It is within the scope of the present invention that the composition, and more particularly the pharmaceutical composition, may comprise one or more of the above-mentioned biologically active compounds, which may be comprised in the composition according to the present invention, as a pharmaceutically active compound and as a further component, respectively. It will be appreciated by the person skilled in the art that in principle any compound may be used as pharmaceutically active compound. Such pharmaceutically active compounds are generally targeted to target molecules involved in the pathology of the disease. Due to the general design principles and modes of action underlying a variety of biologically active compounds, and thus pharmaceutically active compounds, as with any aspect of the present invention, it is possible to apply to virtually any target. Thus, the compounds according to the invention and the respective compositions containing the same may be used for the treatment or prevention of any disease or disease condition for which a biologically active compound of this kind may be used, prevented and/or treated. It is to be understood that in addition to these biologically active compounds, any other biologically active compound may also be part of the composition according to any embodiment of the present invention. Preferably such further biologically active compound comprises at least one negative charge, preferably under conditions where such further biologically active compound interacts or coordinates with the compound according to the invention, more preferably the compound according to the invention is present as a cationic lipid.

As used herein, a biologically active compound is preferably any compound that is biologically active, preferably exhibiting any biological, chemical and/or physical effect on a biological system. Such biological system is preferably any biochemical reaction, any cell, preferably any animal cell, more preferably any vertebrate cell and most preferably any mammalian cell, including, but not limited to, any human cell, any tissue, any organ and any organism. Any such organism is preferably selected from the group comprising mouse, rat, guinea pig, rabbit, cat, dog, sheep, pig, goat, cow, horse, poultry, monkey and human.

It is also within the present invention that any composition according to the present invention, more specifically any pharmaceutical composition according to the present invention, may comprise any additional pharmaceutically active compound.

The compositions according to the invention, in particular pharmaceutical compositions, can be used in a variety of administration forms, of which local and systemic administration are particularly preferred. More preferred is a route of administration selected from the group comprising intramuscular, transdermal, subcutaneous, intravenous and pulmonary administration. As preferably used herein, topical administration means that the respective compositions are administered in a closed spatial relationship to the cells, tissues and organs to which the compositions and biologically active compounds are to be administered, respectively. As used herein, systemic administration refers to administration other than topical administration, and more preferably is administered separately into bodily fluids such as blood and fluids which deliver the composition separately to the cells, tissues and organs to which the composition and biologically active compound are to be delivered separately.

As used herein, cells for trans-cellular membrane transfer of a biologically active compound by means of a compound and composition according to the invention, respectively, are preferably eukaryotic cells, more preferably vertebrate cells and even more preferably mammalian cells. Most preferably, the cell is a human cell.

Any pharmaceutical agents that can be manufactured using the compounds and compositions according to the invention are for the treatment and prevention, respectively, of a patient. Preferably such patient is a vertebrate, more preferably a mammal and even more preferably such mammal is selected from the group comprising mouse, rat, dog, cat, guinea pig, rabbit, sheep, pig, goat, cow, horse, poultry, monkey and human. In a further aspect, the compounds and compositions according to the invention may be used as transfer reagents, more preferably as transfection reagents.

As preferably used herein, a transfer agent is any agent suitable for transferring a compound, more preferably a biologically active compound such as a pharmaceutically active compound, across a membrane, preferably a cell membrane and more preferably transferring such a compound into a cell as described herein above. More preferably, such transfer also includes release from any endosomes/liposomes.

In still further aspects, the invention relates to methods of transfer, more specifically to transfecting cells with a biologically active compound. In the first step, wherein the order of the steps is not necessarily limited, and in particular not limited to the order of the steps described hereinafter, the cells and the membrane and the cells are provided separately. In a second step, the compound according to the invention is provided, together with a biologically active compound such as a pharmaceutically active compound. The reaction can be brought into contact with the cell and the membrane, respectively, and due to the biophysical characteristics of the compounds and compositions according to the invention, the biologically active compound will be transferred from one side of the membrane to the other, or in the case of the membrane forming cells, from outside the cell to inside the cell. It is within the scope of the present invention that the biologically active compound and the compound according to the invention, i.e. the cationic lipid, are contacted before contacting the cell and the membrane, respectively, whereby preferably a complex is formed and such a complex is contacted with the cell and the membrane, respectively.

In a further aspect of the invention, the method of transferring the biologically active compound and the pharmaceutically active compound, respectively, comprises the steps of providing the cell and the membrane, respectively, providing the composition according to the invention and contacting the composition and the cell and the membrane, respectively. It is within the scope of the invention that the composition may be formed before and during contact with the cell and membrane, respectively.

In one embodiment of any of the methods for transferring a biologically active compound as disclosed herein, the method may comprise further steps, preferably a step of detecting whether the biologically active compound has been transferred. This detection reaction strongly depends on the kind of biologically active compound delivered according to the method and will be apparent to the skilled person. It is within the scope of the invention that such methods be performed on any of the cells, tissues, organs, and organisms described herein.

It will be appreciated that in further embodiments, as described herein, a shielding agent is bound to the lipid component of the lipid composition according to the invention, preferably to the cationic lipid.

The invention is further illustrated by reference to the following figures and examples, which can achieve further features, embodiments and advantages of the invention. More specifically, the present invention is to provide a novel,

FIG. 1 shows a basic design of a cationic lipid according to the present invention;

figure 2 shows the synthesis of N-oleyl-palmitylamine as a possible starting material for the synthesis of the compounds according to the invention, wherein such synthesis is according to one of the prior art as described in US 6,395,713;

FIG. 3 depicts the synthesis of N-oleyl-palmitylamine as an important starting material in accordance with the present invention;

figures 4-9 depict the synthesis of β -arginyl-2, 3-aminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride, β -arginyl-2, 3-aminopropionic acid-N-lauryl-N-myristyl-amide trihydrochloride, and e-arginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride;

FIG. 10 depicts the synthesis of an alternative cationic head group as an alternative component for cationic lipid synthesis according to the present invention;

FIG. 11 depicts an alternative synthetic route to β -arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride synthesis;

FIGS. 12A and 12B depict the size distribution and the effect of extrusion and high pressure homogenization, respectively, of a lipid dosage form according to the present invention; and

FIGS. 13A and 13B depict the results of Western Blot analysis and the effect of different concentrations of PEG-substituted lipids.

Example 1: synthesis of N-oleyl-palmitylamine according to the prior art

N-oleyl-palmitylamine is an important starting material for the compounds according to the invention. N-oleyl-palmitylamine may in principle be synthesized as described in US 6,395,713. The respective synthetic schemes are depicted in fig. 2. However, the starting material is technical grade oleyl amide, supplied by e.g. Fluka. Analysis of the starting material by gas chromatography showed a purity of > 70%, with 30% of the starting material consisting of amides with different chain lengths. The reason for this may be that the raw material as such is obtained from a plant source. After reacting the starting materials at 100 to 120 ℃ for 30 minutes, the combination of oleylamine and 1-bromohexadecane (palmityl bromide) gives N-oleyl-palmitylamine. The yield thereof was found to be about 83%.

Example 2: synthesis of N-palmityl-oleylamine according to the invention

With regard to the compounds according to the invention, a new synthesis has been recognized by the inventors of the present invention (fig. 3). This new reaction scheme is based on the discovery by the inventors of the present invention that high amounts of impurities affect the quality of the transfer reagents prepared based on the starting materials. Thus, the reaction was started with oleic acid of 99% purity as shown by gas chromatography and reacted with ethyl chloroformate, TEA and CH₂C1₂Reacted and the reaction thus obtained carboxylic-carbonic anhydride mixed with hexadecylamine (palmitylamine) again having a purity of 99% or more as shown by gas chromatography. The reaction product N-palmityl-oleoyl amide [ #1 [ ]]Followed by reaction with LiAlH₄Reaction (in THF) to give 85% N-palmityl-oleylamine [ #2]]It appeared as a colorless crystalline solid.

More detailed reaction conditions are summarized below.

N-palmityl-oleamide [ #1]Synthesis of (2)

2.62ml (27.5mmol) of ethyl chloroformate are dissolved under inert gas argon in 30ml of anhydrous dichloromethane in a 250ml nitrogen flask according to Schlenk and cooled to 0 ℃. A solution of 7.93ml (25mmol) oleic acid and 4.16ml (30mmol) triethylamine in 40ml dry dichloromethane was added dropwise over 20 minutes under control (stirring). After 30 minutes of conditioning on an ice bath, 50ml of CHCl were added₃A solution of 6.64g (27.5mmol) of palmitylamine in (B) was added dropwise rapidly and the mixture was controlled at room temperature for 2 hours. Subsequently, the solutions were washed three times with 40ml of water each, and the organic phase was washed in Na₂SO₄Dried and the solvent removed using a rotary evaporator. The residue was recrystallized from 100ml of acetone. 11.25g (22.3mmol) of a colorless solid were obtained, corresponding to a yield of 89%.

N-palmityl-oleylamine [ #2]Synthesis of (2)

1M LiAlH in 20ml of ether under an inert atmosphere of argon₄Provided in a 250ml three-necked flask with dropping funnel and reflux condenser, and then a solution of 7.59g (15mmol) of palmityl oleamide in 80ml of THF was added dropwise over 20 minutes. The mixture was refluxed for 2.5 hours, then another 5ml of 1M LiAlH in ether was added₄And refluxed for an additional 2.5 hours. Excess hydride was decomposed with 6M NaOH under ice bath cooling and the precipitate was filtered off. The precipitate was extracted twice each with 40ml of hot MtBE and the combined organic phases were taken up in Na₂SO₄Dried and the solvent removed using a rotary evaporator. The residue was crystallized from 100ml of MtBE at-20 ℃. 6.23g (12.7mmol) of a colorless crystalline solid corresponding to a yield of 85% are obtained.

Example 3: boc-dap (Fmoc) -N-palmityl-N-oleyl-amide [ #3 [ ]]Synthesis of (2)

521mg (1.06mmol) of N-oleyl ene in 10ml of anhydrous dichloromethaneThe base-palmitylamine was dissolved in a 50ml round bottom flask and 289mg (1.17mmol) of EEDQ was added. Subsequently, 500mg (1.17mmol) of Boc-dap (Fmoc) -OH was added under control and the mixture was controlled at room temperature for 20 hours. The solution was transferred with 80ml dichloromethane into a separating funnel and washed three times with 20ml each of 0.1N HCl and with 20ml of saturated NaHCO₃The solution was washed once. In Na₂SO₄After drying, the solvent was removed using a rotary evaporator (fig. 4). A yellowish viscous oil was obtained, which was further purified. R was observed in thin layer chromatography using 1: 1 hexane/ethyl acetate_fIs 0.70.

Example 4: Boc-Dap-N-palmityl-N-oleyl-amide [ #4]Synthesis of (2)

1g of Boc-dap (Fmoc) -N-palmityl-N-oleyl-amide crude product was dissolved in 8ml of anhydrous dichloromethane in a 50ml round bottom flask. 3ml of diethylamine were added and controlled at room temperature (FIG. 4). A thin layer chromatography control of the reaction showed that after 4.5 hours the reaction of the starting product was complete. The volatile components are removed by rotary evaporator and the residue is chromatographed on 40g of silica gel 60(Merck) using 5: 1 hexane/ethyl acetate. The product was eluted using a step gradient consisting of ethyl acetate, 4: 1 ethyl acetate/methanol and 4: 1 dichloromethane/methanol. 576mg (0.85mmol) of Boc-Dap-N-palmityl-N-oleyl-amide were obtained as yellow viscous oil.

Example 5: tetra-Boc- [ beta-arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide][#5] Synthesis of (2)

576mg (0.85mmol) Boc-Dap-N-palmityl-N-oleyl-amide was dissolved in 10ml dry dichloromethane in a 100ml round bottom flask and 210mg (0.85mmol) EEDQ and 403mg (0.85mmol) Boc-Arg (Boc) were added under control₂-OH (FIG. 5). The mixture was controlled at room temperature under an argon atmosphere for 20 hours. Followed byThe dichloromethane was removed by rotary evaporator and the residue in 100ml MtBE was transferred to a separatory funnel. The organic phase was washed with 0.1N HCl, 1N NaOH and saturated NaHCO₃The solution was washed thoroughly in Na₂SO₄Dried and the solvent removed by rotary evaporator. The crude product was then purified by flash chromatography (Combiflash recovery; IscoInc) using hexane/ethyl acetate as eluent. 694mg (0.61mmol) of colorless viscous oil corresponding to a yield of 72% were obtained.

Example 6: beta-arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride [#6]Synthesis of (2)

694mg (0.61mmol) of well-dried tetra-Boc- [ beta-arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide under an argon atmosphere]A25 ml nitrogen flask according to Schlenk was provided and 4N HCl in 8ml dioxane was added (FIG. 5). The mixture is controlled under argon inert gas at room temperature for 24 hours, wherein after about 6 to 8 hours the product precipitates out of solution as an amorphous and partly as a waxy solid. In the reaction (using 65: 25: 4 CHCl)₃/MeOH/NH₄Thin layer control of OH) was completed, any volatile components were removed under high vacuum. 489mg (0.58mmol) of beta-arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide are obtained as the trihydrochloride.

Example 7: n-lauryl-myristylamide [ #7]Synthesis of (2)

18.54g (100mmol) of dodecylamine (laurylamine) and 6.36g (60mmol) of Na were added₂CO₃And 50mg of tetrabutylammonium iodide (TBAI) were suspended in 100ml of anhydrous DMF in a 500ml 3-necked flask with reflux condenser and dropping funnel. Over 110 minutes at 100 ℃, a solution of 16.4ml (60mmol) of 1-bromotetradecane in 100ml of anhydrous dioxane was added dropwise and the mixture was gummed up for a further 3.5 hours at 100 ℃ (fig. 6). Will dissolveThe liquid is filtered at as high a temperature as possible. The precipitate was allowed to settle at 4 ℃ overnight, the crystalline solid was removed and washed with a small amount of cold methanol. The solid was then recrystallized from 200ml of methanol. 9g of colorless leaf-like crystals recrystallized from 100ml of MtBE were obtained. The crystals precipitated at-18 ℃ were blotted dry from the cooled frit and washed with cold MtBE. 7.94g (21mmol) of a colorless crystalline solid are obtained, corresponding to a yield of 35%.

Example 8: boc-dap (Fmoc) -N-lauryl-N-myristylamide [ #8 [ ]]Synthesis of (2)

715mg (1.68mmol) of Boc-dap (Fmoc) -OH was dissolved in 15ml of anhydrous dichloromethane in a 50ml round-bottomed flask and 420mg (1.7mmol) of EEDQ was added. The mixture was kept at room temperature for 45 minutes and then a solution of 641mg (1.68mmol) of N-lauryl-myristylamine in 25ml of anhydrous dichloromethane was slowly added dropwise over 60 minutes (FIG. 6). After a reaction time of 20 hours, the solvent was removed with a rotary evaporator and the residue was transferred with 100ml MtBE to a separatory funnel. The solution was washed with 0.1N HCl and saturated NaHCO₃The solution was washed thoroughly over Na₂SO₄The organic phase was dried and the solvent was removed with a rotary evaporator. Purification by flash chromatography (Combiflash recovery; Isco Inc.) using hexane/ethyl acetate as solvent gave 1.02g of crude product. 607mg of pure product are obtained as a colorless, very viscous oil. Thin layer chromatography with 1: 1 hexane/ethyl acetate afforded R_fIs 0.58.

Example 9: Boc-Dap-N-lauryl-N-myristylamide [ #9 [ ]]Synthesis of (2)

607mg Boc-dap (Fmoc) -N-lauryl-N-myristylamide was dissolved in 8ml dry dichloromethane in a 50ml round bottom flask (FIG. 6). 3ml of diethylamine were added and the reaction was controlled at room temperature for 4.5 hours. The volatile constituents are removed by means of a rotary evaporator and the residue is purified by chromatography using 40g of silica gel 60(Merck) with 5: 1 hexane/ethyl acetate. The product was eluted with a step gradient consisting of ethyl acetate, dichloromethane and 3: 1 dichloromethane/methanol. 372mg (0.655mmol) Boc-Dap-N-lauryl-N-myristylamide was obtained as a pale yellow viscous oil.

Example 10: tetra-Boc- [ beta-arginyl-2, 3-diaminopropionic acid-N-lauryl-N-myristylamide] [#10]Synthesis of (2)

372mg (0.655mmol) Boc-Dap-N-lauryl-N-myristylamide was dissolved in 8ml dry dichloromethane in a 50ml round bottom flask and 162mg (0.655mmol) EEDQ and 311mg (0.655mmol) Boc-Arg- (Boc) were added under control₂-OH (FIG. 7). The mixture was allowed to stand at room temperature for 20 hours. Subsequently, the dichloromethane was removed using a rotary evaporator and the residue was transferred to a separating funnel with 80ml MtBE. The organic phase was washed with 0.1N HCl, 1N NaOH and saturated NaHCO₃The solution was washed thoroughly over Na₂SO₄Dried and the solvent removed on a rotary evaporator. The crude product was purified by flash chromatography (Combiflash recovery; Isco Inc.) using a hexane/ethyl acetate step gradient. 500mg (0.5mmol) of a colorless viscous oil are obtained, corresponding to a yield of 76%.

Example 11: beta-arginyl-2, 3-diaminopropionic acid-N-lauryl-N-myristylamide trihydrochloride Object [ #11]Synthesis of (2)

511mg (0.5mmol) of well-dried tetra-Boc- [ beta-arginyl-2, 3-diaminopropionic acid-N-lauryl-N-myristylamide]A 25ml argon flask according to Schlenk was provided under argon and 10ml 4N HCl in dioxane was added (fig. 7). The mixture was controlled at room temperature under an inert atmosphere of argon for 24 hours, wherein after 6 to 8 hours the product precipitated from solution as a partially amorphous, partially waxy solid. At the completion of the reaction (using 65: 25: 4 CHCl)₃/MeOH/NH₄Thin layer chromatography control of OH) In this case, all volatile constituents are removed under high vacuum. 323mg (0.5mmol) of beta-arginyl-2, 3-diaminopropionic acid-N-lauryl-N-myristylamide are obtained in the form of the trihydrochloride.

Example 12: Boc-Lys (Fmoc) -N-lauryl-N-myristamide [ #12 [ ]]Synthesis of (2)

937mg (2mmol) Boc-Lys (Fmoc) -OH was dissolved in 10ml dry dichloromethane in a 50ml round bottom flask and 495mg (2mmol) EEDQ was added (FIG. 8). The mixture was controlled at room temperature for 60 minutes and then a solution of 764mg (2mmol) of N-lauryl-myristylamine in 30ml of anhydrous dichloromethane was slowly added dropwise over 120 minutes. After a reaction time of 20 hours, the solvent was removed with a rotary evaporator and the residue was transferred with 100ml MtBE to a separatory funnel. The solution was washed with 0.1N HCl and saturated NaHCO₃The solution was washed thoroughly over Na₂SO₄The organic phase was dried and the solvent was removed with a rotary evaporator. Purification by flash chromatography (Combiflash recovery; IscoInc.) using 4: 1 hexane/ethyl acetate as solvent gave 1.757g of crude product. 1.377g of pure product were obtained as a colourless, very viscous oil. Thin layer chromatography with 1: 1 hexane/ethyl acetate afforded R_fIs 0.57.

Example 13: Boc-Lys-N-lauryl-N-myristylamide [ #13]Synthesis of (2)

1.377g Boc-Lys (Fmoc) -N-lauryl-N-myristyl-amide was dissolved in 16ml dry dichloromethane in a 50ml round bottom flask. 6ml of diethylamine were added and the mixture was controlled at room temperature for 5 hours (FIG. 8). Volatile constituents are removed by means of a rotary evaporator and the residue is purified by chromatography using 40g of silica gel 60(Merck) with 5: 1 hexane/ethyl acetate. The product was eluted using a step gradient consisting of ethyl acetate, dichloromethane and 3: 1 dichloromethane/methanol. 556mg (0.911mmol) Boc-Lys-N-lauryl-N-myristylamide and also 119mg of the combined fractions were obtained as a pale yellow viscous oil.

Example 14: tetra-Boc- [ epsilon-arginyl-lysine-N-lauryl-N-myristyl amide][#14]Is/are as follows Synthesis of

556mg (0.911mmol) Boc-Lys-N-lauryl-N-myristyl-amide are dissolved in 40ml dry dichloromethane and 226mg (0.911mmol) EEDQ and 433mg (0.911mmol) Boc-Arg (Boc) are added under control₂-OH (FIG. 9). The mixture was controlled at room temperature for 20 hours. Subsequently, dichloromethane was removed using a rotary evaporator and the residue was transferred to a separatory funnel using 80ml MtBE. With 0.1N HCl and saturated NaHCO₃The organic phase is washed thoroughly with the solution and passed over Na₂SO₄Dried and the solvent removed using a rotary evaporator. The crude product was then purified by flash chromatography (combiflash recovery; Isco Inc.) using an ethane/ethyl acetate step gradient. A colorless viscous oil was obtained in a yield corresponding to 75% of 730mg (0.684 mmol).

Example 15: Epsilon-arginyl-lysine-N-lauryl-N-myristylamide trihydrochloride [ #15] Synthesis of (2)

730mg (0.684mmol) of well-dried tetra-Boc- [ epsilon-arginyl-lysine-N-lauryl-N-myristylamide]A 25ml argon flask according to Schlenk was provided under argon and 10ml 4N HCl in dioxane was added (fig. 9). The mixture was controlled under argon inert gas at room temperature for 24 hours, whereupon, after about 8 hours, the product precipitated from the solution as an amorphous, partially waxy solid. In a process such as that described by using 65: 25: 4 CHCl₃/MeOH/NH₄When the OH thin-layer chromatography-controlled reaction is complete, all volatile constituents are removed under high vacuum. 491mg (0.633mmol) of ε -arginyl-lysine-N-lauryl-N-myristylamide as the trihydrochloride were obtained.

Example 16: tri-Boc-gamma-ureido-alpha, gamma-diaminobutyric acid [ #16 [ ]]Synthesis of (2)

1.31g (6mmol) of Boc-Dab-OH was provided in 15ml acetonitrile in a 100ml round bottom flask and 12mmol Diisopropylethylamine (DIPEA) was added (FIG. 10). Water was then added dropwise until a portion of Boc-Dab-OH was dissolved and then 1.96g (5mmol) of 1, 3-di-Boc-2- (trifluoromethanesulfonyl) guanidine was added. The mixture was controlled at room temperature for 12 hours, whereupon acetonitrile was removed using a rotary evaporator. The water residue was diluted with 5ml of water and 50ml of dichloromethane were added. The reaction was acidified to pH2 by the addition of 2N HCl under control and followed by separation of the organic phase. The aqueous phase was extracted with 50ml dichloromethane and the combined organic phases were subsequently washed with some diluted HCl and saturated NaCl solution. Through Na₂SO₄The organic phase was dried and the solvent was removed using a rotary evaporator. The residue was purified by chromatography on silica gel 60 using 2: 1 hexane/ethyl acetate. 1.138g (2.47mmol) of a colorless amorphous solid corresponding to a 50% yield are obtained.

Example 17: beta-arginyl-2, 3-diaminopropionic acid-M-palmityl-N-oleyl-amide trihydrochloride Substance [ # #6]Synthesis of (2)

1.225g (6mmole) of BOC-Dap-OH in 15ml of complete CH2Cl2 were suspended under an argon atmosphere in a 250ml Schlenk flask containing a funnel and 1.72ml of trimethylamine were added. A solution of 1.52ml (12mmole) of TMSCI in 30ml of complete CH2Cl2 was added dropwise over 15 to 20 minutes at room temperature with vigorous stirring. At the same time, 941mg (5.8mmole) of carbonyldiimidazole were dissolved under an argon atmosphere in 8ml of complete CH2Cl2 in a 100ml Schlenk flask. A solution of 2.66g (5.6 mmoles) of Boc-Arg (Boc)2-OH in 25ml of complete CH2Cl2 was added dropwise at room temperature under stirring over 15 to 20 minutes. Both reaction solutions were stirred at room temperature for 4 h. Subsequently, 832 μ l (6mmole) of triethylamine was added to the first solution at room temperature under an argon atmosphere and the second solution was added dropwise through a dropping funnel over 15 to 20 minutes. After 15 to 20 minutes, 30ml of water are added, stirred vigorously for 45 minutes and the solution is adjusted to pH 2. The organic phase was separated and extracted several times with CH2Cl 2. The combined organic phases were dried with a saturated solution of NaCl and sodium sulfate and the solvent was removed using a rotary evaporator. The glassy residue was purified by flash chromatography on silica gel using dichloromethane as eluent. 2.74g (4.15 mmole; 74%) of a colorless amorphous solid [ Compound 17] are obtained.

This solid was reacted with oleyl palmitylamine [ #2] under substantially similar conditions as in example 10, with the temperature set to 35 to 40 ℃ (yield 72%). Upon cleaving the Boc protecting group as described in example 11, the desired final product, β -arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride, [ #6] was obtained. The product thus obtained can be further purified by flash chromatography on RP-18 silica gel with MeOH/water as eluent.

Example 18: production of Complex consisting of cationic Liposome and siRNA (lipid Complex)

Lipid complexes consisting of cationic Liposomes and siRNA are made using standard techniques known in the art, such as lipid membranes/cakes, ethanol injection procedure, reverse phase evaporation and detergent dialysis procedure [ refer to lipomes as Tools in Basic Research and Industry; jean r. philippot and francis Schuber; CRC Press January 1995 and Liposome Technology: preparation of Liposomes: 001 Gregory Gregoriadis CRC Press I Llc. april1984 ].

The liposomes thus obtained, also referred to herein as lipoplexes, comprise as lipid β -arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride and additionally either 1, 2-dipentanoyl-sn-trioxy-3-diphosphoethanolamine or 1, 2-dioleyl-sn-trioxy-3-diphosphoethanolamine, with 1, 2-dipentanoyl-sn-trioxy-3-diphosphoethanolamine being preferably used. The lipid fractions of the liposomes and lipid complexes are 50 mole% β -arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride and either 50 mole% 1, 2-diphytanoyl-sn-trioxy-3-phosphoethanolamine or 50 mole% 1, 2-dioleenyl-sn-trioxy-3-phosphoethanolamine, respectively.

The combination of 50 mole% of β -arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride and 50 mole% of 1, 2-diphytanoyl-sn-trioxy-3-phosphoethanolamine is also referred to herein as atuFect.

It will be appreciated that in principle any other lipids and lipid compositions disclosed herein may be manufactured using the above-described techniques with further processing steps.

The liposomes and lipoplexes are subjected to further processing steps, respectively, in order to adjust them according to size, polydispersity and layer design. These characteristics can be adjusted by sonication, e.g. extrusion through a porous membrane, and homogenization, preferably high pressure homogenization.

The liposomes thus formed were characterized by photon correlation spectroscopy with a Beckman-Coulter N5 submicron particle analyzer, and such liposomes sized either by extrusion or by high pressure homogenization are depicted in fig. 12A and 12B, respectively.

As can be taken from fig. 12A, the size distribution of the liposomes can be modified with different membranes with different size exclusion, in this case 1,000nm and 400nm, respectively. In both cases, the extrusion step was repeated 21 times. However, it is within the present invention that the size exclusion may be from about 50 to 5000nm and the extrusion step may be repeated 10 to 50 times.

As can be taken from fig. 12B, high pressure homogenization is also a suitable method to modify the size distribution of the liposomes, wherein the size of the liposomes depends on the number of homogenization cycles of said liposomes when such high pressure homogenization is applied. Typical pressures are in the range of 100-.

Example 19: lipid composition and PEG content

To test the effect of PEG on transfection efficacy and delivery of lipid compositions comprising β -arginyl-2, 3-diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride (cationic lipid) as the first lipid component, 1, 2-diphytanoyl-sn-trioxy-3-phosphoethanolamine (DPhyPE) as the first helper lipid and ceramide conjugated to PEG2000(C8mPEG2000) and PEG5000(C8mPEG5000), respectively, the following dosage forms were produced according to the methods disclosed herein:

A₁-A₅the preparation formulation is as follows:

	cationic lipid [ mol%]	DPhypE [ mol% ]]	C8mPEG2000[ mol%]
				A1	50 mol%	49	1
A2	50 mol%	47.5	2.5
				A3	50 mol%	45.0	5.0
A4	50 mol%	42.5	7.5

A5

50 mol%

40.0

10

B₁-B₅Dosage forms

	Cationic lipid [ mol% ]]	DPhypE [ mol% ]]	C8mPEG2000[ mol%]
				B1	49	50 mol%	1
B2	47.5	50 mol%	2.5
				B3	45.0	50 mol%	5.0
B4	42.5	50 mol%	7.5
				B5	40	50 mol%	10.0

For any of the above dosage forms, the lipid concentration was 1.445mg/ml and the siRNA concentration was 15. mu.M in 300mM sucrose. Dilution of the concentrated formed stock complexes produced final siRNA concentrations in cell culture media of 20, 10, 5 nM.

The RNAi molecules contained in the dosage form were directed against PTEN and the sequences were as follows: a first strand having the sequence: u. ofaucacggug-P; having the sequence caccgugaua second strand of a-P, wherein the modification is that the bold printed nucleotide is a 2' -P-methyl nucleotide; the 3' end of any chain starts with a phosphate, as described by P in the above sequence.

The lipid formulations were administered to HeLa cells contained in 6-well plates each containing 40,000 cells/well. PTEN expression was analyzed for cells and the results are depicted in figure 13 as WesternBlots. P110a expression was used as a loading control and detected with antibody. As can be taken from any of the Western Blots depicted in fig. 13A and 13B, the content of PEG compounds, i.e., shielding compounds, can be increased by up to 5 and 7.5 mol%, where the concentration can be higher or can be at the higher end of the range in the case of PEG2000 compared to PEG5000 used.

There the PEG is not removable from the lipid composition compared to those containing the composition, which is a clear advantage. Lipid dosage forms of similar compositions comprising 1, 2-distearoyl-sn-trioxy-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG2000) in the range of 1 to 5 mole% instead of ceramide PEG conjugates can only be incorporated 1 to 2 mole% in order to provide an effective knock down (knockdown).

The features of the invention disclosed in the specification, the claims and/or the drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims (65)

1. A lipid composition comprising

At least one first lipid component selected from the group consisting of,

at least one first helper lipid, and

a shielding compound removable from the lipid composition under in vivo conditions, wherein the first lipid component is a compound according to formula (I),

wherein R is₁And R₂Each and independently selected from the group comprising alkyl groups and alkenyl groups containing from 8 to 30 carbon atoms;

n is any integer between 1 and 4;

R₃is an acyl group selected from the group comprising lysyl, ornithyl, 2, 4-diaminobutyryl, histidyl and an acyl moiety according to formula (II),

wherein m is any integer of 1 to 3, wherein NH in formula (II)₃ ⁺Optionally is absent, and

Y^-is a pharmaceutically acceptable anion, and is a pharmaceutically acceptable anion,

wherein the shielding compound is PEG, and wherein the shielding compound is conjugated to a ceramide, wherein the ceramide comprises at least one short carbon chain substituent of 6 to 10 carbon atoms.

2. A lipid composition according to claim 1, wherein the ceramide is covalently bound to the shielding compound.

3. A lipid composition according to claim 2, wherein the shielding compound is PEG2000 or PEG 5000.

4. The lipid composition according to claim 1, wherein the composition comprises a further component and/or a second helper lipid, wherein the further component is selected from the group comprising peptides and nucleic acids.

5. The lipid composition according to claim 1, wherein the composition comprises a further component and/or a second helper lipid, wherein the further component is selected from the group comprising proteins, oligonucleotides and polynucleotides.

6. The lipid composition according to claim 4, wherein the lipid composition comprises a nucleic acid as an additional component.

7. The lipid composition according to claim 6, wherein the nucleic acid is selected from the group comprising RNAi, siRNA, siNA, antisense nucleic acids, ribozymes, aptamers and spiegelmers, wherein the RNAi has the same design as siRNA and comprises 50 or more nucleotides or base pairs.

8. A lipid composition according to claim 1, wherein the ceramide comprises 8 carbon atoms.

9. A lipid composition according to claim 4, wherein the ceramide is the first helper lipid.

10. A lipid composition according to claim 4, wherein the ceramide is the second helper lipid.

11. A lipid composition according to claim 1, wherein R₁And R₂Each and independently selected from the group comprising lauryl, myristyl, palmitoyl, and oleyl.

12. A lipid composition according to claim 1, wherein R₁Is lauryl and R₂Is myristyl; or R₁Is palmitoyl and R₂Is oleyl.

13. The lipid composition according to claim 1, wherein m is 1 or 2.

14. The lipid composition according to claim 1, wherein the compound is a cationic lipid.

15. A lipid composition according to claim 14, wherein the compound is conjugated with an anion Y^-And (4) associating.

16. A lipid composition according to claim 1, wherein Y is^-Selected from the group comprising halides, acetates and trifluoroacetates.

17. The lipid composition according to claim 1, wherein the compound according to formula (I) is selected from the group comprising:

-beta-arginyl-2, 3-diaminopropionic acid-N-palmitoyl-N-oleyl-amide trihydrochloride

-beta-arginyl-2, 3-diaminopropionic acid-N-lauryl-N-myristyl-amide trihydrochloride

And

-epsilon-arginyl-lysine-N-lauryl-N-myristyl-amide trihydrochloride

18. A lipid composition according to claim 1, wherein the composition comprises a carrier.

19. A pharmaceutical composition comprising a composition according to claim 1 and a pharmaceutically active compound.

20. A pharmaceutical composition comprising a composition according to claim 4 and a pharmaceutically active compound.

21. The pharmaceutical composition according to any one of claims 19 and 20, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

22. The composition according to claim 20, wherein the pharmaceutically active compound and/or the further component is selected from the group comprising peptides and nucleic acids.

23. The composition according to claim 20, wherein the pharmaceutically active compound and/or the further component is selected from the group comprising proteins, oligonucleotides and polynucleotides.

24. The composition according to claim 23, wherein the protein is an antibody.

25. The composition according to claim 23, wherein the protein is a monoclonal antibody.

26. The composition according to claim 22, wherein the nucleic acid is selected from the group comprising DNA, RNA, PNA and LNA.

27. The composition according to claim 22, wherein said nucleic acid is a functional nucleic acid selected from the group comprising RNAi, siRNA, siNA, antisense nucleic acids, ribozymes, aptamers, and spiegelmers.

28. The composition according to claim 4, wherein the first helper lipid and/or the second helper lipid is selected from the group comprising phospholipids and steroids.

29. The composition according to claim 28, wherein the first and/or second helper lipid is selected from the group comprising 1, 2-diphytanoyl-sn-trioxy-3-diphosphoxyphosphoethanolamine and 1, 2-dioleyl-sn-trioxy-3-diphosphoxyphosphoethanolamine.

30. A composition according to claim 28, wherein the content of the first and/or second helper lipid is from 20 to 80 mol% of the total lipid content of the composition.

31. A composition according to claim 30, wherein the content of the first and/or second helper lipid is from 35 mol% to 65 mol%.

32. The composition according to claim 29, wherein the first lipid component is β -arginyl-2, 3-diaminopropionic acid-N-palmitoyl-N-oleyl-amide trihydrochloride and the first and/or second helper lipid is 1, 2-diphytanoyl-sn-trioxy-3-phosphoethanolamine.

33. A composition according to claim 32, wherein the first lipid component is 50 mol% of the total lipid content of the composition and the first and/or second helper lipid is 50 mol% of the total lipid content of the composition.

34. The composition according to claim 4, wherein the first and/or second helper lipid comprises a group selected from the group consisting of a PEG moiety, a HEG moiety, a polyhydroxyethyl starch (polyHES) moiety, and a polypropylene moiety.

35. The composition according to claim 34, wherein the moiety provides a molecular weight of about 500 to 10000 Da.

36. The composition according to claim 34, wherein the moiety provides a molecular weight of about 2000 to 5000 Da.

37. The composition according to claim 34, wherein the helper lipid comprising a PEG moiety is selected from the group comprising 1, 2-distearoyl-sn-trioxy-3-phosphoethanolamine and 1, 2-dialkyl-sn-trioxy-3-phosphoethanolamine.

38. The composition according to claim 37, wherein the PEG moiety of the helper lipid has a molecular weight of 2,000 to 5,000 Da.

39. The composition according to claim 38, wherein said composition comprises as said lipid component β -arginyl-2, 3-diaminopropionic acid-N-palmitoyl-N-oleyl-amide trihydrochloride, 1, 2-diphytanoyl-sn-trioxy-3-phosphoethanolamine as a first helper lipid and 1, 2-distearoyl-sn-trioxy-3-phosphoethanolamine-PEG 2000 as a second helper lipid.

40. The composition according to claim 38, wherein the second helper lipid is present in an amount between about 0.05 mole% and 4.9 mole%.

41. The composition according to claim 40, wherein the second helper lipid is present in an amount of about 1 to 2 mole%.

42. The composition according to claim 1, wherein the composition contains about 1 to 10 mole% of a conjugate of PEG and ceramide.

43. The composition according to claim 1, wherein the composition contains 1 to 7.5 mole% of a conjugate of PEG and ceramide.

44. The composition according to claim 1, wherein the composition contains 1 to 5 mole% of a conjugate of PEG and ceramide.

45. The composition according to claim 42, wherein the ceramide is C8 and PEG is PEG2000 and wherein the content of conjugate of PEG and ceramide is about 1 to 7.5 mole%.

46. The composition according to claim 42, wherein the ceramide is C8 and PEG is PEG5000 and wherein the content of conjugate of PEG and ceramide is about 1 to 5 mole%.

47. The composition according to claim 1, wherein the first lipid component is present in an amount of about 42.5 to 50 mole% and the first helper lipid is present in an amount of about 42.5 to 50 mole%, wherein the sum of the amounts of the first helper lipid and the conjugate of PEG and ceramide and of the first lipid component is 100 mole%.

48. The composition of claim 27, wherein the functional nucleic acid is a double-stranded ribonucleic acid.

49. The composition according to claim 20, wherein the composition further comprises a nucleic acid, wherein the nucleic acid is selected from the group comprising RNAi, siRNA, siNA, antisense nucleic acids, and ribozymes.

50. The composition of claim 14, wherein the composition further comprises a nucleic acid, wherein the nucleic acid is RNAi, wherein the molar ratio of RNAi to cationic lipid is about 0 to 0.075.

51. The composition of claim 14, wherein the composition further comprises a nucleic acid, wherein the nucleic acid is RNAi, wherein the molar ratio of RNAi to cationic lipid is about 0.02 to 0.05.

52. The composition of claim 14, wherein the composition further comprises a nucleic acid, wherein the nucleic acid is RNAi, wherein the molar ratio of RNAi to cationic lipid is about 0.02 to 0.037.

53. Composition according to any one of claims 1 and 4, wherein the first lipid component and/or the at least one helper lipid and/or the shielding compound is present as a dispersion in an aqueous medium.

54. The composition according to any one of claims 1 and 4, wherein the first lipid component and/or the at least one helper lipid and/or the shielding compound is present as a solution in a water-miscible solvent.

55. A composition according to claim 54, wherein the solvent is selected from the group comprising ethanol and tert-butanol.

56. Use of a composition according to any one of claims 1 to 55 for the manufacture of a medicament.

57. Use of a composition according to any one of claims 1 to 55 for the preparation of a transfer agent.

58. The use according to claim 57, wherein the transfer agent transfers the pharmaceutically active component and/or the further component into the cell.

59. The use according to claim 58, wherein the cell is a mammalian cell or a human cell.

60. Use of a composition according to any one of claims 1 to 55, wherein the composition comprises a pharmaceutically active compound and/or a further component, in the manufacture of a reagent for transferring the pharmaceutically active compound and/or the further component into a provided cell or across a provided membrane; and a composition according to any one of claims 1 to 55 for use in contact with said cell or said membrane.

61. The use according to claim 60, wherein the pharmaceutically active compound and/or the further component is capable of being detected in the cell and/or on the membrane.

62. An in vitro method for transferring a pharmaceutically active compound and/or a further component into a cell or across a membrane, said method comprising the steps of:

-providing said cell or said membrane;

-providing a composition according to any one of claims 1 to 55, wherein the composition comprises a pharmaceutically active compound and/or additional components; and

-contacting said cell or said membrane with a composition according to any one of claims 1 to 55.

63. The method of claim 62, wherein the membrane is a cell membrane.

64. A method according to claim 62, wherein the method comprises the further step of:

detecting the pharmaceutically active compound and/or the further component in the cell and/or on the membrane.

65. A lipoplex comprising a lipid composition according to any one of claims 1 to 55; and siRNA.