WO2010009065A2

WO2010009065A2 - Amphipathic peptide compositions

Info

Publication number: WO2010009065A2
Application number: PCT/US2009/050443
Authority: WO
Inventors: Jeremy Baryza; Andrew Geall; Sushma Kommareddy; Jennifer Philips
Original assignee: Novartis Ag
Priority date: 2008-07-15
Filing date: 2009-07-14
Publication date: 2010-01-21
Also published as: WO2010009065A3; WO2010009065A9

Abstract

The present application pertains to a composition, comprising - amphipathic peptides; - lipids and - at least one target compound. Respective compositions are suitable for target compound transport and delivery, for example for systemic or local delivery to a mammal. Also provided are pharmaceutical compositions, comprising respective compositions.

Description

ORGANIC COMPOUNDS

FIELD OF THE INVENTION

The present technology pertains to amphipathic peptide compositions and their use for the delivery and transportation of target compounds.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is an evolutionary conserved biological process for specific silencing of gene expression. Synthetic small interfering (si) RNAs have emerged as an important tool for post-transcriptional gene silencing in mammalian cells in life animals owing to their unique properties, such as potency, specificity and lack of an interferon response. Delivery of RNAi mediating compounds remains a large obstacle for in vivo application of RNAi mediating compounds, including the use as therapeutics following systemic administration. Delivery of siRNAs across plasma membranes in vivo has been achieved using vector based delivery systems, high pressure intravenous injections of siRNA and chemically modified siRNAs, including cholesterol-conjugated, lipid-encapsulated and antibody-linked siRNAs.

Also the delivery of other target compounds such as for example DNA, antisense molecules or small molecules such as pharmaceutically active chemical compounds is a challenge.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to compositions comprising amphipathic peptides and lipids for the transport of target compounds. In one embodiment, the composition comprises amphipathic peptides, lipids and at least one target compound.

It is the object underlying the present application to provide an efficient system for target compound transport, in particular for systemic or local delivery. The amphipathic peptides form particles by solubilising lipids within the composition. These particles may be employed to transport target compounds including, for example, RNA, DNA, small non-coding RNA, RNAi mediating compounds, siRNA compounds, oligonucleotides, antisense molecules, peptides, polypeptides or small molecules.

In another embodiment, the present composition comprises target compounds, wherein the target compound is at least one pharmaceutically active compound. In one embodiment, the present compositions comprise amphipathic peptides, wherein the peptide is modified to alter the physical properties of the particles. The peptides may be modified, for example, by biotmylation, fluorination or the conjugation of binding molecules such as antibodies or fragments.

In another embodiment, the present composition comprises a targeting ligand for the delivery of the composition to the target of choice, for example, to a specific body compartment, organ or tumor.

The present invention further embodies a composition comprising amphipathic peptides and lipids employed as a carrier for at least one target compound

The present invention also provides a method for producing a composition comprising amphipathic peptides, lipids and at least one target compound, wherein the lipids are mixed with the amphipathic peptides and processed to form particles and the particles are contacted with at least one target compound.

Other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

BRIEF DESCRIPTION QF THE FIGURES

Figures 1a to c illustrates three different ways to combine the desired elements, in order to attach the target compound to the particles formed of the amphipathic peptides and lipids. The target compound used in the illustration is a siRNA compound, which is accordingly negatively charged.

In Figure 1a, a lipophilic anchor is attached via a linker group to the siRNA compound. The lipophilic anchor inserts into the phospholipids, thereby anchoring the siRNA to the particles. According to the shown embodiment, the particle also comprises a targeting ligand. Said targeting ligand also comprises a lipophilic anchor The lipophilic anchor is attached via a linker group to the targeting ligand and thus anchors the targeting ligand to the particles by insertion into the phospholipids.

Figure 1b shows an alternative wherein cationic lipids are used as capturing agents in order to associate the siRNA to the particles. The cationic lipids comprise a lipophilic anchor and a cationic head. The lipophilic anchor inserts into the phospholipids, the cationic head is accessible at the surface. The charged groups at the cationic head interact due to the charges with a negatively charged siRNA, thereby associating the siRNA to the particles. According to the shown embodiment, a targeting ligand is directly coupled to the siRNA compound. A respective targeting ligand may be present or may not be present It can be coupled for example by a linker structure to the siRNA.

Figure 1c shows an embodiment, wherein again a lipophilic anchor is used in order to anchor the siRNA to the lipid layer. In the shown embodiment, a targeting ligand is associated with the amphipathic peptides. For example, the targeting ligand can be coupled to lysine residues present in the amphipathic helix. Also more than one targeting ligand can be used.

Figure 2 shows an embodiment for functionalizing the amphipathic peptides. An amphipathic peptide is shown, wherein the lysine side chains are available and thus accessible for chemical modification. The lysine side chains are modified with an alkyne and thus provide an anchoring site for attaching a targeting ligand TL.

Figure 3 shows lipidated targeting motifs useful for particle targeting.

Figure 4 shows an overlay of size exclusion chromatograms of particles made at a peptide to lipid molar ratio of 1:1.75, 1 :3 and 1 :7 and peptide alone at a concentration of 2 mg/ml using peptide of Sequence ID No.1 and lipid POPC. About 200 μl of particles in normal saline were injected onto a superpose 6 column using 50 mM sodium phosphate with 150 mM sodium chloride at 0.5 ml/min as elution buffer with run times up to 40 ml elution volumes. The chromatograms shows UV absorbance at 215 nm plotted against elution volume. Figure 4 demonstrates that the size of the particles varies, depending on the used peptide to lipid molar ratio. An increase of lipids renders bigger particles. From the peptide to lipid molar ratios tested, 1:1.75 provided the smallest particles that had a size of less than 10nm. A respective size exclusion chromatogram also allows the determination of the stokes diameter, when an appropriate standard is used. An alternative method for determining the diameter of the particles is Dynamic light scattering and the same trends are observed.

Figure 5 shows the size exclusion chromatograms of cholesterol conjugated siRNA (a) followed by particles loaded with cholesterol conjugated siRNA at a peptide to lipid molar ratio of 1 :1.75, 1:3 and 1 :7 (b, c, and d) and particles alone at respective molar ratios (e, f and g) with the absorbance reported at 215, 254 and 280 nm. All the particles were made using peptide Sequence ID No.1 and lipid POPC. The samples were prepared in normal saline and about 200 μl of them were injected onto a superpose 6 column using 50 mM sodium phosphate with 150 mM sodium chloride at 0.5 ml/min as elution buffer with run times up to 40 ml elution volumes. As can be derived from the respective figures, loading of the siRNA molecule does not considerably increase the size of the particles. It is expected that less than 10 siRNA, less than 5 and most likely between 1 to 3 siRNAs is/are loaded onto one particle. The number/amount of associated siRNA may also be varied by using different concentrations of siRNAs.

Figure 6 shows a size exclusion chromatogram of particles made using peptide Sequence ID No.1 and lipid POPC at peptide to lipid molar ratio of 1 : 1.75. About 200 μl of particles at a peptide concentration of 8mg/ml were injected onto a superpose 6 column using 50 mM sodium phosphate with 150 mM sodium chloride at 0.5 ml/min as elution buffer with run times up to 40 ml elution volumes. Fractions of 0.5 ml each were collected in a 96-well plate in series of rows throughout the run and are plotted along with the elution volumes against absorbance at 215 nm on the chromatogram. The fractions from C₉-Dg were pooled and concentrated by tangential flow filtration using MicroKros hollow fibers (Spectrum Labs) made of polysulfone with 50 KD cut-off.

Figure 7 shows 2-dimensional NMR spectra (NOESY Spectra) of particles made at a peptide to lipid molar ratio of 1 :1.75 in 5 mM potassium phosphate (KH₂PO₄) buffer made in 90 % v/v H₂O and 10 % v/v D₂O at pH 6.23, 37 ⁰C. The particles with peptide at a concentration of 2 mg/ml of peptide Sequence ID No.1 were used to collect data on Bruker-Biospin NMR at 600 MHz. Figure 9 shows an enlarged section of Figure 7 (of the left upper corner).

Figure 8 shows the structure and proton assignment of the lipid POPC by nuclear magnetic resonance (NMR). A lipid film is made by evaporating off excess methanol from the stock solution of POPC in methanol. A lipid solution or liposomes of POPC were made by hydrating the lipid film with deuterated methylene chloride at a concentration of 1 mg/ml.

Figure 9 shows 2-dimensional NMR spectra (NOESY Spectra) of particles made using peptide Sequence ID No.1 and lipid POPC. The right picture is an enlarged view of the left upper corner from Figure 7. The x-dimension {6-9 ppm) represents proton signals of aromatic amino acids and the y-dimension (0-5 ppm) represents proton signals of lipid and side chains of aromatic amino acids. The particles used were made at a peptide to lipid molar ratio of 1 :1.75 in 5 mM potassium phosphate (KH₂PO₄) buffer made in 90 % v/v H₂O and 10 % v/v D₂O at pH 6.23, 37 ⁰C. The particles with peptide at a concentration of 2 mg/ml were used to collect data on Bruker-Biospin NMR at 600 MHz. In sum, Figures 7 to 9 demonstrate that the peptides have a helical structure in the particles according to the present invention, that the helical peptides interact with the lipids on a molecular level at a defined space and that the particles have a defined structure.

Figure 10 shows the energy changes of cholesterol conjugates siRNA loading onto particles, (a) Microcalorimetry data of 7.31 mg/ml of chol-siRNA titrated into particle at peptide to lipid molar ratio of 1 :1.75 (0.399 mg/ml peptide Sequence ID. No 1 and 0.199 mg/ml of POPC) following subtraction of control titration of cholesterol-siRNA into saline, (b) Schematic representation of loading of cholesterol-si RNA (hypothetically existing as micelles) into lipoprotein particles. The control includes dilution of cholesterol-siRNA which is the de-micellization event. The subtraction of the control from the observed energy changes of cholesterol-siRNA into particles results in energy associated with loading of cholesterol-siRNA into particles. All the energy changes were plotted against molar ratio of cholesterol-siRNA to peptide in particles. Figure 10 shows that the energy profile changes when siRNA is added to the buffer, maybe they form micellar structures. Upon adding the siRNA to the particles, again a change in the energy profile is observed allowing the conclusion that the siRNA and the particles interact, thereby forming a loaded particle carrying at least one target compound.

Figure 11 shows the size exclusion chromatograms of particles along with human lipoproteins. The particles with peptide Sequence ID No.1 and lipid POPC were used at a peptide to lipid molar ratio of 1 :1.75. The chromatograms show injection overlay of (a) particles at a peptide concentration of 2 mg/ml, high density lipoproteins (HDL) at 1 mg/ml and a mixture of HDL and particles (0.5 and 1 mg/ml respectively), (b) particles at a peptide concentration of 2 mg/ml, low density lipoproteins (LDL) at 1 mg/ml and a mixture of LDL and particles (0.5 and 1 mg/ml respectively), and (c) particles at a peptide concentration of 2 mg/ml, very low density lipoproteins (VLDL) at 0.877 mg/ml and a mixture of VLDL and particles (0.438 and 1 mg/ml respectively). Figure 11 shows again the remarkable stability of the particles according to the present application.

Even when mixing the particles according to the present invention (NLPP - Nano Lipid Peptide Particles) with natural lipoproteins such as HDL, LDL and VLDL, and thus with natural lipoproteins, the NLPPs remain as a distinct fraction. Hence, the particles do not interact with the natural lipoproteins or form aggregates or disintegrate under the tested conditions. Accordingly, they are stable in the presence of other lipoproteins. This is an important characteristic for pharmaceutical applications and it also enables efficient targeting.

Figure 12 shows differential scanning calorimetry of peptide and particles. The polts show melting curves of (a) peptide Sequence ID No.1 at 1.11 mg/ml and (b) particles made from peptide Sequence ID No.1 and lipid POPC at peptide to lipid molar ratios of 1 :1.75, 1 :3, and 1:7 at peptide concentrations of 1.11 mg/ml in particles. AH samples of peptide and particles were made in normal saline. This figure shows that the particles have a rather high melting point of more than 80°C and even around respectively above 9O⁰C. This stability is important for an industrial large scale production and for the handling of the particles.

Figure 13 shows florescence microscopy images of particles in hepatocyte cells (Huh7) cells at 24 h of transfection. Particles were made at a peptide to lipid ratio of 1:7 using rhodamine-peptide Sequence ID No.1 and lipid POPC. Alexa488 labelled cholesterol- siRNA with chemistry as described in reference (Wolfrum et al, 2007) was used to load the particles at a concentration of 0.5 mg/ml with peptide at 1 mg/ml in particles. A reverse transfection was performed with Huh7 cells with particles containing cholesterol-siRNA at 500 nM concentration. Following 24 h of transfection the cells were washed of the formulation, fixed and imaged under a florescence microscope. Figure A shows the phase contrast image of the cells, figure B shows the image of the celts taken in the red channel (signal from rhodmine-peptide) and indicates cytoplasmic punctuate staining. Figure C shows the image taken in the green channel (alexa 488- siRNA) and indicates punctuate cytoplasmic staining. Figure D shows the image taken in the blue channel (alexa 488-siRNA) and indicates DAPI stained nuclei. The images in all these channels were merged to study the co-localization of the florescent labels. The results show that the paiictes and the loaded siRNA travel together. The siRNA is stably associated to the particles. The DAPI stained results also show that the siRNA does not enter the nucleus which is important for RNAi.

DETAILED DESCRIPTION QF THE INVENTION

According to one embodiment of the present application, a composition is provided, comprising

- amphipathic peptides;

- lipids; and

- at least one target compound.

The amphipathic peptides used for creating the composition of the present application may be of the same kind or may comprise peptides of a different kind, e.g. of a different amino acid sequence. The peptides can be composed of L and/or D amino acids and may comprise natural as well as non-natural amino acids and amino acid analogues.

The peptides may have an amino acid chain length of less than 100, 50, 35 or less than 30 amino acids. The amphipathic peptides used according to the teaching of the present application solubilise the lipids and form particles. The respectively formed particles may comprise a core of lipids, wherein the helices of the amphipathic peptides are assembled in a belt-like fashion around the lipid core, thereby shielding the hydrophobic parts of the lipids. The shape of the particles may resemble a disc. As respective particles can be synthetically produced, they are of a defined composition and size. They can also be scaled up to large quantities and can be produced with a uniform size, thereby depicting significant advantages over natural lipoproteins. Furthermore, it was shown in extensive experiments that the particles according to the present application are remarkably stable and can be purified and processed {e.g. sterile filtered). These are important advantages for an industrial production process. Furthermore, it was shown that the particles according to the present application are also stable in the presence of natural lipoproteins such as HDL, VLDL or LDL. This is an important advantage for in vivo applications as undesired aggregations or interactions with natural lipoproteins are avoided.

The respective particles comprising amphipathic peptides and lipids can be loaded with at least one target compound in order to form the composition. Hence, the respective particles are suitable as carriers/vehicles for transporting and hence delivery of target compounds. Respective compositions are in particular suitable for delivering at least one target compound to a recipient, e.g. a mammal and in particular a human.

The respective particles formed have a synthetic structure similar to that of known lipoproteins, such as HDL. An important advantage over natural HDL is that the particles according to the present application can be synthetically produced at a large scale. They have a defined composition and are also stable under various conditions. Their defined composition and stability profile make them particularly suitable for pharmaceutical applications.

It was found advantageous to use an amphipathic peptide that is capable to mimic properties of apolipoprotein A1. Apolipoproteins are lipid-binding proteins that are divided into 6 major classes (A, B, C, D, E and H) and several sub-classes. Apolipoproteins in lipoproteins are classified into exchangeable (apo A-I₁ A-Il, A-IV, C-I, C-Il, C-III and E) and non-exchangeable apolipoproteins (apo B-100 and B-48). They are synthesized in the liver and intestine. The exchangeable apolipoproteins are capable of exchange between different lipoprotein particles during lipid metabolism. Structurally, these exchangeable apolipoproteins contain different classes of amphipathic helices-class A (sub-classes A1, A2 and A4), class Y and class G₁ which impart lipid affinity to apolipoproteins. An arπphipathic helix contains hydrophilic amino acids on the polar face and hydrophobic amino acids on the non-polar face. The distribution and clustering of charged amino acid residues in the polar face of the helix is the predominant difference among different classes of amphipathic helices. The design and synthesis of respective peptides that are capable of mimicking the properties of apolipoprotein A1 is known in the prior art, please refer for example to Mishra et al. "Interaction of Model Class A1, Class A2, and Class Y Amphipathic Helical Peptides with Membranes", Biochemistry 1996, August 27; 35{34):11210-20, herein incorporated fully by reference. Furthermore, respective synthetic peptide analogs are known which are able to mimic the lipid- binding and Lecithin-Cholesterol Acetyltransferase (LCAT) activation properties of apolipoproteins. Amphipathic peptides of varying lengths have been designed by various researches for optimum alpha helicity, lipid-binding and LCAT activation.

Suitable amphipathic peptides that can be used according to the present invention are e.g. described in Mishra VK, Anantharamaiah GM, Segrest JP, Palgunachari MN₁ Chaddha M, Sham SW₁ Krishna NR. Association of a model class A (apolipoprotein) amphipathic alpha helical peptide with lipid: high resolution NMR studies of peptide lipid discoidal complexes. J Biol Chem. 2006 Mar 10;281(10):6511-9; Mishra VK, Palgunachari MN. Interaction of model class A1, class A2, and class Y amphipathic helical peptides with membranes. Biochemistry. 1996 Aug 27;35(34): 11210-20;

Anantharamaiah GM.Synthetic peptide analogs of apolipoproteins. Methods Enzymol. 1986; 128:627-47; Navab M, Anantharamaiah GM, Reddy ST, Hama S, Hough G, Grijalva VR₁ Yu N, Ansell BJ, Datta G, Garber DW, Fogelman AM. Apolipoprotein A-I mimetic peptides. Arterioscler Thromb Vase Biol. 2005 JuI; 25(7) 1325-31 ; Navab et al. "Apolipoprotein A - 1 mimetic peptides and their role in athereosclerosis prevention" Nature Clinical Practise October 2006 VoI 3 No. 10; herein incorporated by reference.

According to one embodiment, the amphipathic peptide used in the composition according to the present invention forms a class A amphipathic alpha helix.

The amphipathic peptides used according to the present application can be selected from a group of peptides comprising the following amino acid sequences:

i. DWLKAFYDKVAEKLKEAFLA (Sequence ID. No 1 ) ii. ELLEKWKEALAALAEKLK (Sequence ID. No. 2) iii. FWLKAFYDKVAEKLKEAF (Sequence ID. No. 3) iv. DWLKAFYDKVAEKLKEAFRLTRKRGLKLA (Sequence ID. No. 4) v. DWLKAFYDKVAEKLKEAF (Sequence ID. No. 5); vi. Functional analogs or fragments of the peptides according to i to v, capable of forming a class A amphipathic alpha helix. Particularly advantageous peptides are peptides comprising or consisting of Sequence ID. No. 5 or 1.

Peptide mimetics of apo A1 usualiy do not show any sequence homology to that of apo A-1 but are capable of forming a class A amphipathic alpha helix similar to apo A-1 and also show lipoprotein binding properties similar to that of apolipoproteins. The respective peptides have the ability to solubilise lipids and form particles with the lipids. According to one embodiment the amphipathic peptides show no sequence homology to apo A-1 or other lipoproteins.

According to one embodiment, at least one of the end groups of the peptides is blocked and hence either the N or the C - terminus. At least one of the termini may be acetylated and/or amidated. It was shown, that blocking at least one end group may increase the helical content of the peptide by removing the stabilizing interactions of the helix macrodipole with the charged termini. According to one embodiment, the N- terminal end is acetylated and the C-terminai end is amidated. According to one embodiment, the peptide Ac- Sequence ID. No. 1 - Nh^or Ac - Sequence ID. No. 5 - NH₂ is used.

Furthermore, the peptides can be chemically modified for example in order to alter the physical properties of the particles. Such modifications can be done for example in order to target the particles, to increase their stability, to visualize them in vitro or in vivo, or to alter their distribution patterns, among other effects. Such modifications can be used alone or in combination with one or more other changes to achieve the desired effects. Examples of modifications include but are not limited to biotinylation, pegylated, fluorination and the conjugation of binding molecules such as antibodies or fragments thereof.

Modifying groups can be attached e.g. to either the C or N terminus or the side chains of the amino acids of the peptides with or without a linker of various lengths and compositions. It is also within the scope of the present application to modify appropriate side chains of the amino acids. Such modifying groups could be composed of small molecules, peptides, carbohydrates, antibodies or fragments thereof, aptameres, polymers or other molecular architectures.

The introduction of these modifications can be made by any method known in the prior art. For example, modified amino acids could be used in the synthesis of the peptide chain. Such unnatural amino acids could contain the entire desired modification, or a functionality such as a free or protected thiol group for use in forming disulphide bonds or adding into unsaturated systems, in azide or alkyne for use in cycloaddition chemistry, or an additional amino or carbonyl group for use in condensation reactions any of which could be used to introduce an extra functionality later in the synthesis.

The modifications may also be made to the peptides to increase the stability or improve the physical properties of the particles. Such modifications can include, but are not limited to, multimerisation of the peptide motifs by linking one of the ends of two or more peptides together, for cross-linking of peptides by linking sidechains of natural or non-natural amino acids of one or more peptides together. Such connections can be made by linkers of various lengths and compositions. Multimerisation of the peptides can be accomplished as part of the peptide synthesis or by reacting functional groups on the peptide, such as side chains e.g. of lysine, acid side chains, amino termini, or unnatural amino acids comprising an appropriate functionality, with bifunctional or multi-functional liners such as activated diacids, diamines or other compatible functional groups.

The lipids used in the present invention can also be of the same or different kind. The lipid that is used in the composition of the present application may have at least one of the subsequent characteristics: (1) it is selected from the group consisting of triglycerides, phospholipids, cholesterol esters and cholesterol; (2) it is a neutral lipid; {3) it is a phospholipid; and/or (4) it is selected from the group consisting of POPC, DMPC, DOPC, DPPC and sphingomyelin.

The lipid may be selected from the group consisting of triglycerides, phospholipids, cholesterol esters and cholesterol. The respective lipids are also found in lipoproteins of the human plasma. Lipoproteins are divided into four major classes - chylomicrons, very low density lipoproteins, low density lipoproteins and high density proteins, which vary in size and compositions. Triglycerides, phospholipids, cholesterol esters and cholesterol are the major lipids present in the respective lipoproteins. It is advantageous to use endogenous lipids in order to reduce the toxicity.

It is advantageous to use a neutral lipid. The lipid may be selected from the group consisting of POPC, DMPC, DOPC, DPPC and sphingomyelin. Also other lipids can be used as long as they are able to form particles with the amphipathic peptides.

According to one embodiment, approximately 16 amphipathic peptides per particle form a double band around the lipid core comprising approximately 54 lipids. Of course, depending on the components and size of the particles, the amounts may vary.

According to one embodiment, the peptide to lipid molar ratio lies between 1 :1 and 1 :10 and more advantageously between 1 : 1.75 to 1 :7. It was found that the size of the particles varies depending on the chosen peptide to lipid molar ratio. The more lipids are used, the bigger the particles get. For obtaining rather smalt particles having a size of less than about 25nm it is advantageous to use a peptide to lipid molar ration of less than 1:2. Particularly advantageous results were achieved with a ratio of about 1 : 1.75.

It is advantageous that the particles formed by the amphipathic peptides and lipids have a size of less than 500 nm, less than 450nm, less than 400nm, less than 350 nm, less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm and less than 100 nm. For pharmaceutical applications it is in particular advantageous that the particles have a size of less than 50, 35, 30, 25, 20 and even less than 10nm. To use rather small particles having a size of less than 25 or even less than 10nm is advantageous as they show a good systematic distribution to all body compartments and should thus also facilitate a target specific uptake. The use of larger particles often causes problems regarding toxicity and distribution patterns that are related to the size of the particles. The small lipid/peptide particles provided in the present applications avoid respective problems due to their structure and size. The respective particles can be loaded with the at least one target compound to be delivered. To use rather small particles is also advantageous in case targeting of the particles to specific body compartments or cells or receptors is intended. Using small particles, e.g. having a size of less than 50 and preferably less than 20nm, enables more efficient targeting of the particles.

In order for at least one target compound to be delivered, it must be associated with the respective particles formed by the amphipathic peptides and the lipids. There are several possibilities to achieve a respective association. According to one embodiment, the target compound comprises a lipophilic anchor. The lipophilic anchor can be for example directly attached to the target compound or a linker can be used in order to allow attachment of the lipophilic anchor. The lipophilic anchor inserts into the lipid core of the particle, thereby anchoring the target compound via the lipophilic anchor to the particle formed by the amphipathic peptides and the lipids.

According to an alternative embodiment, the target compound is associated with the particles by charge interactions. For example, in case a negatively charged target compound is to be delivered, a positively charged capturing agent can be used in order to capture and associate the negatively charged target compound to the particle. The respective capturing agent may e.g. comprise a lipophilic anchor, which allows anchoring of the capture agent to the particle via the lipophilic anchor which inserts into the lipid core. The capturing agent according to this embodiment would comprise cationic groups and can be for example a cationic lipid. The charged groups are available for interaction with the negatively charged target compound when the capturing agent is anchored to the particle via the lipophilic anchor. Thereby, an association of the target compound to the particle is achieved. Also combinations of different association principles described are within the scope of the present invention.

The target compound can be of any nature and can be for example selected from the group consisting of RNA, DNA₁ small non-coding RNA, RNAi mediating compounds, in particular siRNA compounds, oligonucleotides, antisense molecules, peptides, polypeptides or small molecules. The present delivery system is particularly useful to deliver RNAi mediating compounds. The RNAi mediating compound is preferably selected from the group consisting of miRNA, siRNA and shRNA. According to one embodiment, the RNAi mediating compound is a siRNA. The siRNA may have a length of less than 35, 30, 27 or 25bp and comprises a double-stranded section. It may have 3' overhangs (e.g. 2bp) or may comprise blunt ends. Usually, it will predominantly comprise RNA nucleotides but may also comprise DNA and non-natural nucleotides in one or both strands.

The target compound may be a pharmaceutically active compound that must be delivered to a mammal, in particular a human e.g. via systemic or local delivery.

According to one embodiment, the amphipathic peptide is capable of binding a target such as e.g. a receptor or a cell surface structure such as a cell marker. E.g. the amphipathic peptides described above which are capable of mimicking properties of apolipoprotein A1 may be able to interact with the SRB-1 receptor and thus are suitable for a targeted delivery to cells carrying the SRB-1 receptor, such as hepatocytes and the adrenal glands. However, in order to be able to provide a targeted delivery of the composition to a target of choice (e.g. a body compartment, an organ, a cell type or a tumor), it is advantageous that the composition comprises a targeting ligand. A respective targeting ligand allows a targeted delivery of the composition to the target of choice, e.g. to a specific body compartment, organ or tumor. Furthermore, the targeting ligand may enable a target specific uptake into the cell. Targeting is in particular advantageous for small particles, having a size of less than 50 nm and in particular in the 5 to 20nm range. Various strategies can be used in order to provide a targeted delivery, such as for example targeting of the folate and asialoglycoprotein receptors, glucosaminoglycans and various receptors and markers expressed on tumor cells through strategies including but not limited to using binding molecules such as antibodies and antibody fragments specific for the respective target, anticalines, aptamers, small molecules, natural and non-natural carbohydrates, peptides and polypeptides as targeting ligands.

The targeting ligand is also associated with the particle(s) formed by the amphipathic peptides and the lipids. Several approaches are conceivable. According to one embodiment, the targeting ligand comprises a lipophilic anchor. The targeting ligand is thus anchored via the respective lipophilic anchor to the particle as the lipophilic anchor inserts into the lipid core. The lipophilic anchor can be directly linked to the targeting ligand or by use of an appropriate linker structure.

According to a further embodiment, the targeting ligand is linked to the target compound. This can be done by direct attachment/coupling or by use of appropriate linker groups. Also non-covalent associations are within the scope of the present application.

According to a further embodiment, the targeting ligand is attached or associated to at least one of the amphipathic peptides. This can be done for example by non-covalent or covalent attachment. Again, an appropriate linker group can be used

Also combinations of the above-described approaches are possible and within the scope of the present invention.

The lipophilic anchors that can be used in order to associate the targeting ligand, the capturing agent and/or the target compound described above, can be for example selected from the group consisting of

- cholesterol

- hydrophobic fatty acids and - bile acid derivatives.

Respective groups have been shown to be useful to achieve anchoring to the lipids of the particles according to the present invention. It has been shown that lipid anchors such as cholesterol are particularly useful for tightly anchoring the target compound, the targeting ligand and/or the capturing agent to the particles. In case a hydrophobic fatty acid is used, it is in particular useful if the lipophilic anchor is strongly hydrophobic and has e.g. a long alkyl and/or alkenyl chain having e.g. at least 18 C atoms. It is also possible to use bile acid derivatives comprising a hydrophobic group. E.g. stearoyl, docosanyl and lithocholeic-oleoy! are suitable lipophilic anchors.

According to one embodiment, the lipophilic anchor is attached to the targeting ligand, the capturing agent and/or the target compound via a cleavable linker which comprises e.g. a disulfide bridge. This embodiment is in particular useful for anchoring the target compound. Preferably, linkers are used which are acid cleavable. This embodiment is particularly useful in case siRNA is delivered as target compound. It is assumed that the siRNA associated via the lipophilic anchor to the particles is contained in the eπdosoms upon entering the target cell. One important aspect for efficient RNA interference is that the siRNA is capable of leaving the endosom. The use of an acid- cleavable linker has the advantage that the linker is cleaved upon entering/processing in the endosom, thereby releasing the target compound. This simplifies the release of the target compound from the carrier particles. This embodiment also enables to use a lipophilic anchor which binds particularly tight to the lipids of the particles. A respective tight anchorage prevents undesired/unintentional detachment of the target compound from the particles.

Examples of acid-labile and biodegradable linkers contain a chemical group such as acetals, ketals, orthoesters, imtnes, hydrazones, oximes, esters, N- alkoxybezylimidazoles, enol ethers, enol esters, enamides, carbonates, maleamates, and others known to those skilled in the art. Another example is linkers containing peptide sequences known to be substrates for proteases.

Also provided with the present invention is a composition, comprising particles of amphipathic peptides and lipids for use as carrier for at least one target compound. Respective particles are in particular useful for delivering at least one target compound to a mammal, in particular a human. Delivery is preferably systemic or local. The details of the respective particles, in particular the nature of the amphipathic peptides, the lipids and the potential use of targeting ligands and anchoring moieties is described in detail above and also applies to the composition according to the present application which can be used for transporting and delivering a target compound. For delivery/transport, the particles comprising the amphipathic peptides and the lipids are mixed with the at least one target compound in order to obtain a composition also comprising the target compound to be delivered.

Also provided according to the present invention is a pharmaceutical composition which comprises a composition as is outlined above. For details regarding the composition, we refer to the above detailed description. The pharmaceutical composition may comprise suitable carriers and diluents.

Also provided is a method for producing a composition according to the present application, comprising amphipathic peptides, lipids and at least one target compound. According to one embodiment, the lipids are mixed with the amphipathic peptides and the respective mixture is then contacted with the target compound in order to load the composition with the target compound. As is outlined above, the lipids form particles with the peptides which are believed to mimic lipoprotein structures. For mixing the lipids with the peptides, the mixture can be vortexed in order to thoroughly mix the lipids with the peptides. In case the peptides and/or the lipids are comprised in alcohol such as methanol, the alcohol should be evaporated after mixing the components. The film formed by the peptides and lipids is dried and is afterwards hydrated with a buffer in order to form disc-like particles comprising the amphipathic peptides and lipids. The respective particles are then contacted with the at least one target compound in order to allow the association of the final particles carrying the target compound.

If the target compound comprises e.g a lipophilic anchor, the respective anchor is according to one embodiment attached to the target compound before the respectively modified compound is contacted with the particles. Attachment of the anchor can be done for example by chemical modification as described above. For certain applications it is particularly advantageous to use a cleavable linker, we refer to our above disclosure. Upon mixing the at least one target compound carrying a lipophilic anchor with the particles comprising the amphipathic peptides and the lipids, the lipophilic anchor of the at least one target compound inserts into the lipid core of the particle, thereby associating the at least one target compound to the particles.

The following Examples are intended to further illustrate the invention and are not to be construed as being limitations thereon.

EXAMPLES

Example 1 - materials used

POPC from Chemi (Basalmo, Italy), DOPC, DMPC and DPPC from Avanti Polar Lipids (Alabaster, AL)₁ Peptides Sequence ID No. 01, Sequence ID No. 03, Sequence ID No. 04 and Sequence ID 05 from American peptide company lnc (Sunnyvale, CA), methanol HPLC grade from Acros (Pittsburgh, PA), DiI from Invitrogen (Carlsbad, CA), Human HDL₁ LDL and VLDL from Millipore Corp. (Bellirica, MA), cholesterol conjugated siRNA are synthesized as described (Wolfrum et al, 2007)

Example 2 - preparation of the particles Stock solutions of peptide and lipid are made in methanol at a concentration of 10 mg/ml. Necessary aliquots of the stocks are transferred to glass vials to obtain peptide to lipid molar ratios ranging from 1:0.877 to 1 :7 (weight ratios of 4:1 to 1 :2). The mixture is vortexed and methanol is evaporated on a rotovap to obtain a clear lipid-peptide film. The particles are obtained by hydrating the film with sterile filtered normal saline at a peptide concentration of 2 mg/ml.

Example 3 - measurement of the particle size by dynamic light scattering

The particles formed by hydration are characterized for particle size by dynamic light scattering on Malvern zetasizer Nano-ZS (Malvern Instruments, Milford MA) at a back scattering angle of 173°. Undiluted particles are used for measurements The peptide of Sequence ID No.1 are used to form particles at peptide to lipid molar ratios of 1:1.75, 1 :3 and 1:7and the size is characterized by dynamic light scattering, number average reported in nm followed by polydispersity index in parenthesis: 5.80 nm (0.1), 15.80 nm (0.15) and 19.92 nm (0.27) respectively with lipid POPC, 8.28 nm (0.4), 11.26 nm (0.16) and 1.72x10⁴ nm (1.0) respectively with lipid DOPC, 10.04 nm (1.0), 17.34 nm (0.76) and 46.14 (0.67) respectively with lipid DPPC, and 5.24 nm (0.15), 5.51 nm (0.16) and 7.12 nm (0.05)respectively with lipid DMPC.

Other amphipathic peptides (Sequence ID No. 2, 3 and 4) are also evaluated for particle formation with lipid POPC. The particles are formed at peptide to lipid molar ratios of 1:1.75, 1:3 and 1 :7, characterized by dynamic light scattering for particle size, number average reported in nm followed by polydispersity index in parenthesis 5.21 nm (0.7), 5.70 nm (0.68) and 27.82 nm (0.63) respectively with peptide Sequence ID No.2, 4.67 nm (0.1), 8.18 nm (0.13) and 5.95x10⁴ nm (1.0) respectively with peptide Sequence ID No.3, and 5.24 nm (0.21), 6.26 nm (0.23) and 8.74 nm (0.57) respectively with peptide Sequence ID No.4

Example 4 - size exclusion chromatography

The particles are sized on Akta explorer 900 (Amersham Biosciences) using superpose-6 column (GE Health Care Life Sciences). The particles are eluted with 50 mM Sodium phosphate with 150 mM sodium chloride at a flow rate of 0.5ml/min for about 40 ml volume. 0.5 ml fractions of the eluent were collected into a 96-well plate (with 8 rows from A to H and 12 columns 1 to12) in a row fashion starting from Ai to Ai₂ followed by row B to H. Data is collected at 215 nm, 254 nm and 280 nm. A mixture of low and high molecular weight gel filtration markers of known stokes diameter are run under similar conditions. The size of the particles are determined by comparing the elution volumes of the samples with that of the standards. The particles are loaded with cholesterol conjugated siRNA (see Wolfrum et al, 2007) and their stokes diameter are determined by size exclusion. About 200 μl of particles are injected on to the size exclusion column at a peptide concentration of 1 mg/ml and siRNA at 0.5 mg/ml.

The particles from peptide Sequence ID No.1 and lipid POPC at peptide to lipid molar ratio of 1:1.75, 1 :3 and 1:7 are prepared and are characterized by size exclusion chromatography (see Figure 4). Elution peak fraction (elution volume in ml), Stokes diameter (in nm) of the particles- at molar ratio of 1 :1.75 the particles are eluted at D₃ (19.39 ml) has a stokes diameter of 2.92 nm, after loading with cholesterol-siRNA they were eluted at C_e-C₇ (15.14 ml) with diameter 8.99 nm. -at molar ratio of 1 :3 the particles were eluted at C₁₂-D₁ (18.14 ml) has a stokes diameter of 4.70 nm, after loading with cholesterol-siRNA they are eluted at C₆ (14.89 ml) with diameter 9.35 nm. -at molar ratio of 1:7 the particles are eluted at C₉ (16.39 ml) has a stokes diameter of 7.20 nm, after loading with cholesterol-siRNA they are eluted at C₄-C₅ (14.14 ml) with diameter 10.42 nm (see Figure 5).

Example 5 - concentration by tangential flow filtration (TFF) Following size exclusion the particles are concentrated using MicroKros hollow fibers (Spectrum Labs). A 50 KD cut off Microkros module is used for this purpose. The luerlok sample ports are connected through a peristalitic pump for continuous flow of the sample through the system. The designated luerlok is connected to the filtrate or waste which is collected. All the connections are made with tubing of smallest diameter in order to reduce the void volumes of the whole system. The whole concentration process is stopped when the volume of the sample is equal to or lower than the void volume and is indicated by the introduction of air bubbles into the system. The MicroKros filter is pre-wetted with normal saline before use.

Particles made from peptide Sequence ID No.1 and lipid POPC at a molar ratio of

1 :1.75 are used. About 200 μl of the particles at a peptide concentration of 8 mg/ml are injected on to a Superose column to perform size exclusion (see Figure 6). The peak fractions Cg-D₉ are combined to give 6.5 ml and were concentrated to 2 ml by TFF. The pooled fractions are characterized by dynamic light scattering for particle size, number average reported in nm are followed by polydispersity index in parenthesis 5.6 nm (0.242), after concentration the particles are found to be at 6.69 nm (0.424) and comparable to the same unprocessed particles before size exclusion chromatography, 5.80 nm (0.1). These data demonstrate that the particles and in particular their size does not change when they are concentrated. The particles are thus remarkably stable and do not form detrimental amounts of aggregates or other artificial products. It is also shown that the particles can be sterile filtered and still remain stable. This is a considerable advantage as it allows the industrial production of the particles having defined characteristics. This is particularly important for pharmaceutical applications.

Example 6 • SEC fraction analysis for peptide and lipid content

The peptide content of the pooled/concentrated fractions is analyzed by UV absorbance at 215 nm. The lipid content is estimated using Phospholipid C reagent (Wako Diagnostics, Japan), a colorimetric enzymatic assay for determination of phospholipids. The absorbance of the chromogen is measured at 600 nm.

About 200 μl of particles made from peptide Sequence ID No.1 and lipid POPC at a molar ratio of 1 : 1.75 is injected on to a superpose column at a peptide concentration of 8 mg/ml (see Figure 6). Following size exclusion the peak fractions C₉-D₉ combine and are concentrated by TFF. The pooled fractions after size exclusion are estimated to have 1.27 mg of the peptide and 0.35 mg of lipid. After TFF the retentate contains 0.92 mg of peptide and 0.35 mg of lipid, and 0.04 mg of peptide is found in the filtrate waste. Example 7 - characterization of particles by NMR

Nuclear overhouser effect spectroscopy (NOESY), 2-D NMR is used to study the peptide-lipid interactions and particles formed are evaluated by 1-D NMR. The peptide-lipid films ware prepared as described and hydrated using 5 mM potassium phosphate (KH₂PO₄) buffer made in 90 % v/v H₂O and 10 % v/v D₂O at pH 6.23, 37 ⁰C. The particles are formed from peptide Sequence ID No.1 and lipid POPC (molar ratio 1:1.75) at a concentration of 2 mg/ml are used to collect data on Bruker-Biospin NMR at 600 MHz.

NOESY uses dipolar interaction of spins to correlate protons, this correlation depends on the distance between protons and a NOE signal is observed only when the distance is less than 5 ⁰A The spectra of particles has NH-NH NOE signals which indicate the interactions of α-proton to α-proton and confirm the helical structure of the peptide (see Figure 7). In the 2-D NMR of particles, the x-axis dimension from 6-9 ppm shows protons from aromatic ring and backbone of the peptide (N-H), and the Y-axis dimension from 0-5 ppm shows signal from protons of lipid and side chains of the peptide. The proton assignment of aromatic amino acids tyrosine (Y - 6.99, 7.22 ppm), and phenylalanine (F - 7.31 ppm), and lipid the double bond linked protons at 4.5 ppm resolved from the rest were known from 1-D NMR. The NOE signals at the intersection of 6.99, 7.22 and 7.31 ppm (on x-dimension) with 4.5 ppm (on Y-dimension) indicate the interactions between the protons of aromatic amino acids with double bond linked protons of the lipid (see Figure 9).

Example 8 - isothermal microcalorimetry

The energy changes associated with siRNA loading on to particles are studied by isothermal titration microcalorimetry (Mirocal ITC). The experiments are carried out at 25 ⁰C by adding 0.28 ml of the lipid-conjugated siRNA (titrant) to 1.4 ml of the particles in solution. The particles at a peptide to lipid ratio of 1 :1.75 are made by hydrating the peptide lipid film with phosphate buffered saline (PBS) and dialyzed with a 3500 Da cut-off dialysis membrane in PBS to get rid of any free peptide. The peptide and lipid content are checked before and after dialysis and particles with peptide at 0.399 mg/ml used for the titration. The cholesterol-conjugated siRNA with chemistry as described (see Wolfrum et al, 2007) at 8.66 mg/ml are used for the titration (see Figure 10).

During this isothermal titration, the energy associated with drop wise addition of the titrant in this case cholesterol-conjugated siRNA is measured. A blank titration of siRNA into buffer is conducted and subtracted from the actual titration of siRNA into particles to get the energy associated with loading of the siRNA into particle. The energy changes are expressed in Kcal/Mole and plotted against molar ratio siRNA to peptide (in particles). The dilution of siRNA into buffer is found to be an endothermic reaction with positive heat of reaction and the titration of siRNA into particles resulted in an exothermic reaction with negative heat of energy.

Example 9 - invitro stability of particles by size exclusion In order to study the stability of particles, the particles are co-incubated in presence of human lipoproteins (HDL, LDL and VLDL) and are characterized by size exclusion chromatography. The particles with peptide to lipid molar ratio of 1:1.75 are used. The particles with a final peptide concentration of 1 mg/ml are incubated with individual lipoproteins at 0.5 mg/ml, and are injected on to the size exclusion column. The particles are found to co-elute along with HDL but are seen to exist as a distinct peak when injected with LDL and VLDL. In both cases, a slight shift in the particle peak is observed (see Figure 11).

Example 10 - invitro stability of particles by differential scanning calorimetry Differential scanning calorimetry is used to study the unfolding events associated with the peptide and particles. This technique is used to measure the amount of heat required to increase the temperature of the sample and reference, resulting in peaks at phase transition temperatures at which more heat is required by the samples to be maintained at the same temperature as the reference. In case of proteins the melting temperatures is determined at which half of the protein exists in an unfolded state.

The peptide of Sequence ID No.1 is used to form particles at peptide to lipid (POPC) molar ratios of 1 :1.75, 1 :3 and 1 :7, the particles with peptide at concentration of 1.11 mg/ml and lipid at 0.55 mg/ml are used. The peptide alone and lipid alone are used as controls and the samples are scanned from 20 ⁰C to 130 ^βC.

The DSC curves are obtained showing (Figure 12) a phase transition of peptide alone at 50 ⁰C and, for particles with peptide to lipid molar ratio at 1:1.75 a phase transition at 105 ⁰C is observed, for particles with peptide to lipid molar ratio at 1 :3 and 1 :7 a phase transition of 93 ⁰C was observed.

Example 11 - cell trafficking by florescence microscopy

Huh7 cells are cultured and split on the day of the experiment. The particles are prepared using peptide of Sequence ID No.1 at a peptide to lipid molar ratio of 1 :7. Out of the total peptide used, 31.25 mole % of it is substituted with rhodamine-labeled peptide of Sequence ID No.1. The particles are then loaded with Alexa488 (green) labeled cholesterol conjugated siRNA at a final concentration of 0.5 mg/ml with peptide in the particles at 1 mg/ml in the final formulation. Reverse transfection of the particles is carried out by co-incubating the cells with particles at siRNA concentrations of 500 nM. At the end of 24 h transfection, the cells are washed to remove free formulation, fixed and are observed under a florescence microscope (see Figure 13). The microscopy pictures show phase contrast image (PCl) of the cells, cells are observed under red channel with punctate staining corresponding to the rhodamine- labeled peptide, cells are observed under green channel with punctate staining corresponding to the Aiexa488 (green) labelled cholesterol-siRNA and DAPI stained cells with nucleus stained in blue channel. Upon merging the above 4-channels co- localization of siRNA (Alexa488 labelled) and peptide (rhodamine labelled) is observed in the cytoplasmic vesicles (image not shown)

Claims

1. A composition, comprising

- amphipathic peptides;

- lipids and

- at least one target compound.

2. The composition according to claim 1 , wherein the amphipathic peptides and the lipids form disc - like particles with a lipid core.

3. The composition according to claim 1 or 2, wherein at least one of said amphipathic peptides has at least one of the following characteristics:

- it forms a class A amphipathic alpha helix;

- it has an amino acid chain length of less than 100, 50, 35 or less than 30; it shows no sequence homology to apolipoprotein A1 ;

- it mimics properties of apolipoprotein A1 ;

- it comprises a class A (apolipoprotein class) motif; - it is selected from the group of peptides comprising at least one of the following amino acid sequence: i. DWLKAFYDKVAEKLKEAFLA (Sequence ID. No 1 ) ii. ELLEKWKEALAALAEKLK (Sequence ID. No. 2) iii. FWLKAFYDKVAEKLKEAF (Sequence ID. No. 3) iv. DWLKAFYDKVAEKLKEAFRLTRKRGLKLA (Sequence ID. No. 4) v. DWLKAFYDKVAEKLKEAF (Sequence ID. No. 5) vi. functional analogs or fragments of i to v capable of forming a class A amphipathic alpha helix;

- it is modified; - it is synthetically produced;

- at least one of the end groups is blocked; and/or

- the C and/or N-terminus is acetylated and/or amidated.

4. The composition according to claims 1 , 2 or 3, wherein the lipid has at least one of the following characteristics:

- it is selected from the group consisting of triglycerides, phospholipids, cholesterol esters and cholesterol;

- it is a neutral lipid; - it is a phospholipid; and/or - it is selected from the group consisting of POPC, DMPC, DOPC₁ DPPC and sphingomyelin.

5. The composition according to any one of claims 1 to 4, wherein the particles of amphipathic peptides and lipids have a size of less than 50nm, less than 25nm or less than 15nm.

6. The composition according to any one of claims 2 to 5, wherein the at least one target compound is associated with a particle formed by the amphipathic peptides and lipids.

7. The composition according to any one of claims 1 to 6, wherein the at least one target compound comprises a lipophilic anchor.

8. The composition according to any one of claims 1 to 7, wherein the at least one target compound is selected from the group consisting of RNA, DNA, small non-coding RNA, RNAi mediating compounds, SiRNA compounds, oligonucleotides, antisense molecules, peptides, polypeptides or small molecules.

9. The composition according to any one of claims 1 to 8, wherein the at least one target compound is a pharmaceutically active compound.

10. The composition according to any one of claims 1 to 9, wherein the amphipathic peptide is capable of binding a target.

11. The composition according to any one of claims 1 to 10, wherein the composition comprises a targeting ligand and/or a capturing agent for associating the target compound.

12. The composition according to claim 11 , wherein

the targeting ligand comprises a lipophilic anchor;

- the capturing agent comprises a lipophilic anchor;

- the targeting ligand is linked to the at least one target compound and/or - the targeting ligand is linked to at least one of the amphipilic peptides.

13. The composition according to claim 7 or 12, wherein the lipophilic anchor is selected from cholesterol and hydrophobic fatty acids.

14. The composition according to at least one of the claims 7, 12 or 13, wherein the lipophilic anchor is attached via a cleavable linker.

15. Use of a composition comprising particles comprising

- amphipathic peptides and - lipids

as a carrier for at least one target compound.

16. The use according to claim 15, for delivering a target compound to a mammal.

17. The use according to claim 15 or 16, wherein the composition has the characteristics as defined in at least one of the claims 2 to 5 and 10 to 14.

18. The use according to at least one of claims 15 to 17, wherein the at least one target compound is selected from the group consisting of RNA, DNA, small non-coding RNA,

RNAi mediating compounds, siRNA compounds, oligonucleotides, antisense molecules, peptides, polypeptides or small molecules

19. The use according to at least one of the claims 15 to 18, wherein the at least one target compound has the characteristics as defined in at least one of the claims 6 to 9.

20. The use according to at least one of the claims 15 to 19, wherein the composition is for systemic or local delivery.

21. A pharmaceutical composition comprising a composition according to any one of the claims 1 to 14.

22. A method for producing a composition according to claims 1 to 14, wherein the lipids are mixed with the amphipathic peptides and processed to form particles, and wherein the particles are contacted with at least one target compound.