ZA200608457B - Lipids, lipid complexes and use thereof - Google Patents

Description

—_—— ++ - Lipids, lipid complexes and use thereof -_—

The present invention is related to cationic lipids, compositions containing the same and use thereof as well as a method for transferring chemical compounds into cells.

Both molecular biology ams well as molecular medicine heavily rely on the introduction off biologically active compeounds into cells. Such biologically active compounds typically~ comprise, among others, DNA, RNA as well as peptides and proteins, respectively. The barrier— which has to be overcomes is typically a lipid bilayer which has a negatively charged outer— surface. In the art, a numrber of technologies have been developed to penetrate the cellular membrane and to thus introduce the biologically active compounds. Some methods conceived for laboratory use, however, 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, would, if at all, only allow a local delivery of biologically active compounds. Apart from said lipid bilayer cellular membwranes also comprise transporter systems. Accordingly, efforts were undertaken to use this kincd of transporter systmes in order to transfer the biologically active compounds across the cell mamembrane. However, due to the specificity or cross-reactivity of such transporter systems, their usee is not a generally applicable method.

A more generally applicabl e approach described in the art for transferring biologically active compounds into cells, is thes use of viral vectors. However, viral vectors can be used only for transferring genes efficiently into some cell types; but they cannot be used to introduce chemically synthesised mole-cules into the cells.

An alternative approach wass the use of so called liposomes (Bamgham, J. Mol. Biol. 13, 238- 252). Liposomes are vesicles which are generated upon association of amphiphilic lipids in water. Liposomes typically comprise concentrically arrangecl bilayers of phospholipids.

Depending on the number o=f layers liposomes can be categorised as small unilamelar vesicles, multilamelar vesicles and large multilamelar vesicles. Liposomes have proven to be effective delivery agents as they allow to incorporate hydrophilic compounds into the aqueous intermediate layers, whereas hydrophobic compounds are incorpowrated into the lipid layers. It is well known in the art that both the composition of the lipid formulation as vevell as its method of preparation “have an effect on the structure and sE ze of the resultant lipid aggregates and thus on the liposomes. Liposomes are also known to incomporate cationic lipids.

Cationic lipdds have, apart from being components of liposomes, also att—racted considerable attention as ~they may as such be used for cellular delivery of biopolymers. UJsing cationic lipids any anionic «compound can be encapsulated essentially in a quantitive manner— due to electrostatic interaction. Xn addition, it is believed that the cationic lipids interact with the negatively charged : cell membramnes initiating cellular membrane transport. It has been found that the use of a liposomal fosrmulation containing cationic lipids or the use of cationic lipids as such together with a biolo-gically active compound requires a heuristic approach as each formulation is of limited use because it typically can deliver plasnmids into some but not all cell types, usually in the absence of serum.

Charge and/or mass ratios of lipids and the biologically active compounds to be transported by them have turned out to be a crucial factor in the delivery of different types Of said biologically active composunds. For example, it has been showesn that lipid formulations swmitable for plasmid delivery comprising 5,000 to 10,000 bases in size, are generally not effective for the delivery of oligonucleoticies such as synthetic ribozymes or amtisense molecules typically comprising about to about 50 bases. In addition, it has recently been indicated that optimal Jelivery conditions for antisense oligonucleotides and ribozymes are different, even in the same ce=11 type.

US patent 6,395,713 discloses cationic lipid basesd compositions which typically consist of a lipophilic gro-up, a linker and a head group and the use of such compositiomns for transferring biologically active compounds into a cell.

The problem underlying the present invention was to provide a meanss for introducing biologically active compounds into cells, preferabl y animal cells. A further pr—oblem underlying the present inwention is to provide a delivery agemt for nucleic acids, particul- arly small nucleic acids such as ssiRNA, siNA and RNAI or aptamers zand spiegelmers.

These problemas are solved by the subject matter of the independent claimss attached hereto.

Preferred embodiments may be taken from the attached claims dependent therecon.

In a first aspect the problem tanderlying the present invention is soL ved by a compound according to formula (I), 0

R1 ns

R2 NH, + o wherein R; and R; are each and independently selected froma the group comprising alkyl; n is any integer betwee=n 1 and 4;

R3 is an acyl selected from the group comprising lysyl, ©mithyl, 2,4-diaminobutyryl, histidyl and an acyl moiety according to formula (IT), oO

H

N NH, m hl .

NH, + NH + v a wherein m is any intege=r from 1 to 3 and

Y is a pharmaceutically~ acceptable anion.

In an embodiment R, and R; -are each and independently selected from the group comprising

Rauryl, myristyl, palmityl and oMeyl.

Mn an embodiment R; is lauryl aand R; is myristyl; or

R; is palmityl and R; is eoleyl.

I'n an embodiment m is 1 or 2.

In an embodiment the compound is a cationic lipid, prefer—ably in association with a-n anion Y".

In an embodiment 7Y is selected from the group comprising halogenids, acetate and trifluoroacetate.

In an embodiment the acompound is selected from the grou_p comprising - B-arginyw~1-2,3-diamino propionic acid-N-pal mityl-N-oleyl-amide trihys/drochloride 2 Va a Fa Fa Fa ra Fa

H,C a

HC om NH, + NH, + HEN

Cr Cr “NEL +

Cl - B-arginyM-2,3-diamino propionic acid-N-laur-yl-N-myristyl-amide trih=ydrochloride

I BA

PC CCOS AARNE

HC = cr’ NE, +

Cr and - g-arginyl—lysine-N-lauryl-N-myristyl-amide £xihydrochloride

0) ul HN

HC NH + ot

POPPY. NH, + HN NH

HC | ct oO

Imm a second aspect the problem underlying the present invention is solved by a compossition comprising as a lipid componert a compound according to the first aspect, and a carrier.

In an embodiment the composition comprises a further constituent .

In a third aspect the problem underlying the present invention ms solved by a pharmaceutical co xmposition comprising a compound according to the first aspect and a pharmaceutically asctive compound and preferably a pharmaceutically acceptable carrier.

In an embodiment of the second and third aspect the pharmaceutically active compound anmd/or thes further constituent is selecte«d from the group comprising pepticies, proteins, oligonucleoti des, poRynucleotides and nucleic acicls.

In an embodiment of the second and third aspect the protein is an antibody, preferabMy a mo=noclonal antibody.

In =an embodiment of the secommd and third aspect the nucleic aci d is selected from the gr-oup commprising DNA, RNA, PNA arad LNA.

In =n embodiment of the second and third aspect the nucleic acid is a functional nucleic aecid, whesteby preferably the functioral nucleic acid is selected from ®he group comprising RNNA|, siRTNA, siNA, antisense nucleic acid, ribozymes, aptamers and spieggelmers.

In awn embodiment of the second and third aspect the composition Further comprises at least sone helper lipid component, wherebwy preferably the helper lipid component is selected from the group comprising phospholipids and steroids.

In a preferred embodiment of the second and third aspect the helper lipid component iss selected from the group comprising 1,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine and 1,2-dioleyl- sn-glycero-3-phosphhoethanolamine.

In an embodiment eof the second and third aspect the content of the belper lipid component is from about 20 mol 24 to about 80 mol % of the overall lipid content of the composition.

In a preferred embwodiment of the second and third aspect the content of the helper lipid component is from asbout 35 mol % to about 65 mol %.

In an embodiment of the second and third aspect the lipid is B-arginyl-2,3-diamino p-Topionic acid-N-palmityl-N-o leyl-amide trihydrochloride, and the helper lipid is 1,2-diphyta—moyl-sn- glycero-3-phosphoetThanolamine.

In a preferred embocliment of the second and third aspect the lipid is 50 mol% and thes helper lipid is 50 mol% of the overall lipid content of the composition.

In an embodiment off the second and third aspect the cormposition contains at least tw helper lipids.

In a preferred embod-iment of the second and third aspect at least one helper lipid comprises a moiety which is selected from the group comprising a PEG moiety, a HEG momicty, a polyhydroxyethyl star—ch (polyHES) moiety and a polypropylene moiety, whereby such moiety preferably provides a -molecule weight from about 500 to 1 0000 Da, more preferably fron about 2000 to 5000 Da.

In a preferred embodi ment of the second and third aspect the helper lipid comprising th .e PEG moiety is selected frorm the group comprising 1,2-distearoy1-sn-glycero-3-phosphoethanol=amine, 1,2-dialkyl-sn-glycero—3-phosphoethanolamine; and Ceram ide-PEG

In a more preferred enmbodiment of the second and third aspect the PEG moiety has a moBecular weight from about 500 Da to 10000 Da, preferably from about 2,000 to 5,000 Da, more preferably a molecular weight of 2,000 Da.

In an even more pref-erred embodiment of the second and th rd aspect the composition ceomprises as the lipid compoonent B-arginyl-2,3-diamino propionic acid-N-palmityl-N-ole—yl-amide trihydrochloride, as aa first helper lipid 1,2-diphytanoyl-sn— glycero-3-phosphoethanolarmnine and as a second helper lipsid 1,2-disteroyl-sn-glycero-3-phosphoe=thanolamine-PEG2000.

In a still more prefer-red embodiment of the second and third aspect the content of thes second helper lipid is from about 0,05mol% to 4,9 mol%, preferablsy about 1 to 3 mol%.

In a still further mores preferred embodiment of the second. and third aspect the contermt of the lipid is from 45 mol%% to 50 mol%, the content of the first helper lipid is from 45 to 5-0 mol% and, under the proviseo that there is a PEGylated second helper lipid, the content of the= second helper lipid is from aWbout 0,1 mol% to about 5 mol %, preferably from about 1 to 4 moI1% and more preferably abou 2 % , whereby the sum of the content of the lipid, of the lipid, of —the first helper lipid and of thes second helper lipid is 100 mol% and whereby the sum of the firs—t helper lipid and the second he=lper lipid is 50 mol%.

In a preferred embodirment of the second and third aspect the «composition contains either a) 50 mol'% of B-arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyH -amide trihydro~chloride, 48 mol%=5 of 1,2-diphytanoyl-sn-glycero-3-phos phoethanolamine; and 2mol% 1,2-distearoyl-sn-glycero-3-phosphoetthanolamine-PEG2000. or b) 50 mol%S of B-L-arginyl-2,3-L-diamino propiomnic acid-N-palmityl-N-oleyl—amide trihydroc=loride, 49 mol%e 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine; and 1 mol% N(Carbonyl-methoxypolyethylenglycc»1-2000)-1,2-distearoyl-sn-gl=ycero- 3-phosph_oethanolamine, preferably the sodium salt thereof.

In a preferred embodiment off the second and third aspect the Functional nucleic acid is a d=ouble- stranded ribonucleic acid, wimerein the composition further conprises a nucleic acid, preferably a functional nucleic acid which is more preferably a double-stranded ribonucleic acid andl most preferably a nucleic acid seliected from the group comprisingg RNAi, siRNA, siNA, ant—isense

Tucleic acid and ribozyme, whereby preferably the molar ratior of RNAi to cationic lipid iss from about 0 to 0.075, preferably from about 0.02 to 0.05 and even moore preferably 0.037.

Mn a preferred embodiment off the second and third aspect the «compound and/or the helper— lipid

Component is present as a dispersion in an aqueous medium.

In a preferred embodiment of ~ the second and third aspect the <ompound and/or the helper lipid

Component is present as a soltation in a water miscible solvent, “whereby preferably the solveent is selected from the group comprising ethanol and tert.-butanol.

Im a preferred embodiment of #the second and third aspect the functional nucleic acid is a dowble- s#randed ribonucleic acid, pre#ferably a nucleic acid selected from the group comprising RNAI,

SERNA, siNA, antisense nucleic acid and ribozyme, and where=by preferably the molar ratio of

RNAI to cationic lipid is from about 0 to 0.075, preferably from about 0.02 to 0.05 and «even nmore preferably 0.037

Im a preferred embodiment of the second and third aspect the composition contains a nucleic acid, whereby the charge ratios of nucleic acid backbone phosphates to cationic lipid nitrogen atoms is about from 1: 1,5 — 7, gpreferably 1: 4. :

In a preferred embodiment off the second and third aspect thhe size of the particles in the dispersion is about 120 nm.

In a preferred embodiment of the second and third aspect the clispersion is a stock dispersion comtaining about 1 to 100 uM s=SRNA, whereby preferably the stock dispersion is diluted in v=ivo or in vitro by 1: 100 to 1:10000, more preferably 1: 1000.

In a fourth aspect the problen underlying the present inventfon is solved by the use o=f a cormpound according to the first aspect or a composition accordimmg to the second or third aspect,

for the manufacature of a medicament, preferably for the treatment ozf cancer and/or cardiovascular related diseases.

In an embodiment of the fourth aspect the medicament is for the treatment of cancer, whereby preferably the can cer is selected from the group coxmprising solid and non-s. olid tumors and whereby more pre=ferably the solid tumor is selected from the group comprising pancreatic cancer, breast canceer, prostate cancer, lung cancer, colon cancer and hepatocellul ar carcinoma.

In an embodiment of the fourth aspect the cancer inwolves a process selected from the group comprising angioge=nesis and neoangiogenesis. : :

In an embodiment of the fourth aspect the medicament is for administering the raucleic acid to a cell selected from the group comprising endothelial cells, epithelial cells arad tumor cells, preferably the cell iss an endothelial cell.

In an embodiment o=f the fourth aspect the endothelial cells are endothelial cells of vasculature.

In an embodimen® of the fourth aspect the vasculature is vasculature arising from neoangiogenesis, pre=ferably tumor associated neoangiog=enesis.

In an embodiment of the fourth aspect the vasculature is selected from the gro up comprising liver vasculature, h_eart vasculature, kidney vasculature, pancreactic vasculature and lung vasculature.

In an embodiment of ~ the fourth aspect the medicament iss for systemic administration.

In an embodiment of the fourth aspect the medicament is for local administration.

In an embodiment off the fourth aspect the medicament is for the treatment of cardiovascular related diseases, whesreby the cardiovascular diseases awe selected from the groump comprising coronary heart diseas e, heart failure, hypertension, thrormbosis, myocardial infarct=ion, ischemic heart diseases such as angina pectoris and arteriosklerosiss.

In an embodim-ent of the fourth aspect the medicament is for the treatment oef angiogenesis related diseases.

Preferably such angiogenesis is related to the following organ _s and diseases where angiogenesis is described as causing such dis ease and, therefore, allowing= for the use of the composition according to the present invention (Carmeliet P., Nature Medicin _e 9, 653 — 660 (2003)): blood vessels vascular malformations, DiGeorge syndrome, HFMT, cavernous hemangioma, arther-osclerosis, transplant arteriopathy, hypertension, diabetes, restenosis adipose tisssue obesity skin psoriasis, warts, allergic dermatitis, scar keloicds, pyogenic granulomas, blistering disease, Kaposi sarcoma in A_IDS patients, hair loss, skin purpura, telangiectasia, venous lake forrmation eye persistent hyperplastic witreous syndrome, diabetic retinopathy, retinopathy of prematurity, choroidal neovascularizaticon lung primary pulmonary hypertension, asthma, nasal polyps, neonatal : respiratory distress, pulmonary fibrosis, emphysema intestines inflammatory bowel and. periodontal disease, ascites, peritoneal adhesions reproductivee system endometriosis, uterine bleeding, ovarian cyst:s, ovarian hyperstimulation, pre-eclampsia bone, joints arthritis, synovitis, osteomyelitis, osteophyte formation, osteoporosis, impaired bora fracture healing nervous system Alzheimer disease, am yotrophic lateral sclerosiss, diabetic neuropathy, stroke gastrointestinal gastric or oral ulcerations, Crohn disease kidmey nephropathy

In a fifth aspect the problem underlying the present invention is solved by the= use of a compound according t-o the first aspect and/or the composition according to the second _ and/or third aspect for the manufacture of a diagnostic agent.

In a sixth aspect the problem underlying the present invention is solved by the use of a compound according to the first aspect or a composition according to the ssecond and/or third aspect, as a transferring agent.

In an embodiment of the sixth aspect the transferring agent transfers a pharmaceutically active component and/or a further constituent into a cell, preferably a mammal—ian cell and more preferably a human cell.

In an embo=diment of the sixth aspect the cell is an endothelial cell, pre—ferably a vascular associated eradothelial cell.

In a seventlm aspect the problem underlying the present invention is solved_ by a method for transferring =a pharmaceutically active compound and/or a further constituent irato a cell or across amembrane, preferably a cell membrane, comprising the following steps: - providing the cell or the membrane; - providing a compound according to any of the first aspect; - providing the pharmaceutically active compound and/or the fumrther constituent; and - contacting the cell or the membrane with the pharmaceutically active compound and/or the further constituent, and the compound according to the= first aspect.

In an eighth aspect the problem underlying the present invention is solved by a method for transferring a pharmaceutically active compound and/or a further Constituent into a cell or across a membrane, preferably a cell mermbrane, providing the following ssteps: - providing the cell or the membrane; - providing a composition according to the second or #third aspect; and - contacting the cell or the membrane with the compossition according to the secon or third aspect.

In an embodiment of the seventh or eighth aspect the pharmaceutically active compouncl comprising as further step: . - detecting the pharm aceutically active compound ancd/or the further constituent inm. the cell and/or beyorad the membrane.

In a ninth aspect the problem underlying the present invention is solved by a method for the synthesis of N-palmityl-oleylamine comprising the following steps: - providing oleic acid; - providing palmitylanine; - reacting the oleic acid and the palmitylamine to fomm N-palmityl-oleoylamide; and - reducing the N-palmityl-oleoylamide to N-palmityl-ole=ylamine, whereby the oleic acid is at least 90 %, more preferably 95 % and —most preferably 99 % pure, whereby the percentage is the molar ratio of oleic acid and any fatty acid different from oleic acid.

- - ~ P ’ = # ~—— y Din iy 7i

AR0 06 Fata

WO 2005/105152 = PCT/EP2005/004=920 13

In an embodiment of the ninth a spect the oleic acid and the palmitylamine are reacted at room temperature.

In an embodiment of the ninth asgpect the oleic acid is subject tos a pre-treatment prior to re=acting it with the palmitylamine, whereby the pre-treatment comprisses reacting the oleic acicl with ethylchloroformate, preferably in anhydrous dichloromethane or anhydrous tetrahydrofuran.

In an embodiment of the ninth aspect the reaction is performed at 0° C, preferably undemr inert gas.

In an embodiment of the ninth aspect the reaction is further reacted with an acid scavenger, whereby the acid scavenger is preferably selected from the g-roup comprising triethyla mine, diisopropylethylamine and pyridin €.

In an embodiment of the ninth asp ect the molar ratio of chlorofomrmic acid ethyl ester, oleic acid, triethylamine and palmitylamine is about 1-1.05:1:1: 1-3 : 1-1. 10. :

In an embodiment of the ninth aspect the reduction of the N-palnityl-oleoylamide to N-palmmity- oleylamine is performed using LiA 1H,.

In an embodiment of the ninth aspect upon reacting the oleic ac=id with the palmitylamine=, the reaction is washed, precipitated ancl the precipitate thus obtained owptionally re-crystallised.

In a tenth aspect the problem urnderlying the present invention is solved by the use of a compound according to the first aspect or a composition accordine g to the second or third asspect for systemic administration, preferably systemic administration to a vertebrate.

In an embodiment of the tenth aspect the vertebrate is a mamma_l, more preferably a man—imal selected from the group comprising -mouse, rat, guinea pig, cat, dos, monkey and man. }

The compounds according to the present invention can, as depicte=d in Fig. 1, be regarded aus to comprise a lipophilic group formed “by the R1-N-R2 moiety, a link—er group formed by the CC 0)-

CH(NH;") (CH,),-NH moiety and a. head group formed by the R3- moiety. The present invemntor bas surprisingly found that this kind of compound exhibiting a posi_tive charge at the linker group is particularly suitable to- transfer biologically” active compounds over a cell membrane and preferably into cells, more preferably animal cells. Also, the present inventor “has surprisingly found that the transfer mediated by the compo unds according to the present iravention will be particularl=y effective if the biologically active compound is a nucleic acid, rmore preferably siRNA andl siNA.

As preferably used herein, the term alkyl refers to a saturated aliphatic radical co ntaining from 8 to 20 carbon atoms, preferably 12 to 18 carbon atoms, or a mono- or polyunsaturated aliphatic hydrocarbosn radical containing from 8 to 30 carbon atoms, containing at least @one double and triple bond_, respectively. Thus, in a preferred embodiment, the term alkyl also commprises alkenyl and alkinyl . Alky refers to both branched and urxbranched, i. e. non-linear or straight chain alkyl groups. Pre=ferred straight chain alkyl groups contain from 8 to 30 carbon atoms. More preferred straight chain alkyl groups contain from 12 to 18 carbon atoms. Preferred branche=d alkyl groups contain frorm 8 to 30 carbon atoms, whereby the number from 8 to 30 carbon ators refers to the number of carbon atoms forming the backbone of such branched alkyl group. The backbone of the branche d alkyl group contains at least one alkyl group as branching off from_ the backbone, with the alkyl group being defined as herein, more preferably with the alkyl grosup comprising short chain alkyl groups, more preferably comprising from 1 to 6, even more prefe=rred 1 to 3 and most preferred 1 C atom. More preferred are branched alkyl groups containing 122 to 18 carbon atoms in thes backbone with the branching alkyl groups being defined as in thes foregoing. A particularly ypreferred alkyl group is the phytanyl group.

In an alterna_tive embodiment, the alkyl is an unsaturated branched or unbranched alkyl group as defined above. More preferably, such unsaturated aliphatic hydrocarbon radical contains 1, 2 or 3 or 4 double bonds, whereby a radical having ome double bond is particularly pmreferred. Most preferred is oleyl which is C18: 1delta, i. e. an aliphatic hydrocarbon radical having 18 C atoms, where=by at position 9 a cis configured double bond is presented rather than a single bond linking C ato m number 9 to C atom number 10.

As used here=in, n is any integer between 1 and 4, which means that n may be 1, 2, 3 and 4. As used herein, rn is any integer between 1 and 3, which means that m maybe 1,2 and 3.

It is to be umderstood that the compounds according to the present invention aare preferably cationic lipids. More preferably, any of the NHI or NH, groups present in thee compounds according to the moresent invention are present in a protonated form. Typically, any positive charge of the compound according to the present invention is compensated byw the presence of an anion. Such anion can be a monovalent or polyvalert anion. Preferred anions are halides, acetate and trifluoroacetat=e. Halides as used herein are woreferably fluorides, chlorides, iodides and bromides. Most preferred are chlorides. Upon association of the catiosmic lipid and the biologically active compound to be transferred into a cell, the halide anion is replaced by the biologically actives compound which preferably exhibits one or several negative charges, although it has to b-€ acknowledged that the overall charge of the biologically exctive compound is not necessarily negative.

It is to be acknowBedged that any compound accomrding to formula (I) comgprises at least two asymmetric C atonms. It is within the present invertion that any possible emnantiomer of such compound is disclo=sed herein, 1. €. in particular the R_-R; S-S; R-S and S-R ena—ntiomer.

The compounds ac- cording to the present inventiora can form a compositioan or be part of a composition, where®by such composition comprises a carrier. In such composition which is also referred to herein as lipid composition the compound=s according to the present invention are also referred to as the liroid component(s). Such carrier is preferably a liquid carriear. Preferred liquid carriers are aqueous carriers and non-aqueous carriers. Preferred aqueous c arriers are water, aqueous buffer systesms, more preferably buffer systems having a physiologic-al buffer strength and physiological samlt concentration(s). Preferred no n-aqueous carriers are so_lvents, preferably organic solvents such as ethanol, tert.-butanol. Without wishing to be bound b-y any theory, any water miscible organic solvent can, in principle, be used. It is to be ackno wledged that the composition, more paarticularly the lipid composition can thus be present as or fom liposomes.

The composition according to the present invention may comprise one or mmore helper lipids which are also referr—ed to herein as helper lipid conmponents. The helper lipidl components are preferably selected f5rom the group comprising phospholipids and steroids. P=hospholipids are preferably di- and monoester of the phosphoric acid. PPreferred members of the Phospholipids are phosphoglycerides amnd sphingolipids. Steroids, as eased herein, are naturally occurring and synthetic compoundls based on the partially hydrogenated cyclopenta [a]phenanthrene.

Preferably, the steroicis contain 21 to 30 C atoms. A particularly preferred steroic is cholesterol.

Particularly preferred helper lipids are 1,2-diphytaanoyl-sn-glycero-3-phosphoe—thanolamine (DPhyPE) and 1,2-diolesoyl-sn-glycero-3-phosphoethancolamine (DOPE).

Particularly preferred - compositions according to the present invention comprise= any of PB- arginyl-2,3-diaminoprogpionic acid-N-palmityl-N-oleyl- amide trihydrochloride [#6] , B-arginyl- 2,3-diaminopropionic acid-N-lauryl-N-myristyl-amide trihydrochloride [#11] or— s-arginyl- lysine-N-lauryl-N-myrisstyl-amide trihydrochloride [#15] in combination with DPhyP®E, whereby the content of DPhyPE is preferably 80 mol %, 65 mol 24, 50 mol % and 35 mol %, wxhereby the term mol % refers to thes percentage of the overall lipid «content of the composition, i— ¢. the lipid content of the composition including the cationic lipid according to the present invention and any additional lipid, includirg, but not limited to, any helper lipid.

It is within the presemmt invention that the compositieon according to the presen=t invention preferably comprises thes compound according to the preesent invention and/or one or several of the helper lipid(s) as di. sclosed herein, whereby either athe compound according to —the present invention, i. e. the catioric lipid, and/or the helper lipid component is present as a di spersion in an aqueous medium. Al ternatively, the compound accoarding to the present inventio-, i. e. the cationic lipid, and/or the= helper lipid component is/are peresent as a solution in a water miscible solvent. As an aqueous medium, preferably any of the aqueous carrier as disclosed herein is used. Preferred water miscible solvents are any solvent which form a homogenous gohase with water in any ratio. Prefer-red solvents are ethanol and tert-.-butanol. It is to be acknowl edged that the composition, more particularly the lipid composition can thus be present as or forms liposomes.

It is to be acknowledged that the composition according to the present invention in Mts various embodiments may 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. Such pharmaceutica 1ly acceptable carrier may, preferably, be selected from the group of carrier as defined hereir in connection with the co—mposition according to the present invention. It will be understood by those skilled in the ar@ that any composition as described herein may, in principle, be also used as a pharmaceutical commposition provided that its ingredients and any combination thereof is pharmaceutically acceptable. A pharmaceutical composition comprises a pharmaceutically active compoun_d. Such pharmaceutically active compound can be the same as the further constituent of the cormposition according to the present invention which is pxeferably any biologically active compound, more preferably amny biologically active compourmd as disclosed herein. Whe further constituent, pharmaceutic=ally active compound and/or bio logically active compounc are preferably selected from the group comprising peptides, proteirs, oligonucleotides, polymmucleotides and nucleic acids.

Preferably, army such biologically active compound is a negatively chargged molecule, The term negatively charged molecule means to inchmde molecules that have _at least one negatively charged group that can ion-pair with the positi-vely charged group of the cationic lipid according to the present invention, although the present i nventor does not wish to te bound by any theory.

In principle, the positive charge at the linker raoiety could also have sorme effect on the overall structure of either the lipid as such or any coamplex formed between th _e cationic lipid and the negatively chmarged molecule, i. e. the biolos gically active compound_ Apart from that, the additional possitive charge introduced into the Lipid according to the preseent invention compared to the cationic= lipids disclosed in US patent 6,3 95,713, should contribute to an increased toxicity of this lipid ass taught by Xu Y, Szoka FC Jr.; Biochemistry; 1996 May (37, 35 (18): 5616-23. In contrast to whaat the one skilled in the art woulld have expected from thiss document of the prior art the compo-unds according to the present iravention are particularly ssuitable for the various purposes discleosed herein and are in particular Jevoid of any increased tox=icity.

A peptide as p-referably used herein is any polymer consisting of at least ®wo amino acids which are covalently~ linked to each other, preferabBy through a peptide bormd. More preferably, a peptide consist=s of two to ten amino acids. A particularly preferred embodiment of the peptide is an oligopeptide= which even more preferably commprises from about 10 to =about 100 amino acids.

Proteins as pre=ferably used herein are polymer.s consisting of a plurality of amino acids which are covalently linked to each other. Preferably ssuch proteins comprise ab-out at least 100 amino acids or amino acid residues.

A preferred protein which may be used in connesction with the cationic lip=id and the composition according to thee present invention, is any antibocly, preferably any monocleonal antibody.

Particularly preferred biologically active compounds, i. e. pharmaceutically active compounds and such furthe=r constituent as used in connection with the composition according to the present invention are nucleic acids. Such nucleic acids can be either DNA, RNA _, PNA or any mixture thereof. More preferably, the nucleic acid is a functional nucleic acid. A functional aucleic acid as preferably used herein is a nucleic acid which is mot a nucleic acid coding for a peptide and protein, respectively. Preferred functional nucleic amcids are siRNA, siNA, RNAi, antisense- nucleic acids, ribozymes, aptamers and spiegelmers which are all known in the art.

SIRNA are small interfering RNA as, for example, described in international patent application

PCT/EP03/08666. These molecules typically consist eof a double-stranded RNA structure which comprises between 15 to 25, preferably 18 to 23 nucle=otide pairs which are base-pairing to each other, i. e. are essentially complementary to each owmther, typically mediated by Wa-tson-Crick base-pairing. Ones strand of this double-stranded RNA_ molecule is essentially complexrmentary to a target nucleic acid, preferably a mRNA, whereas the second strand of said doubl e-stranded

RNA molecule is essentially identical to a stretch of said target nucleic acid. Thhe siRNA molecule may be flanked on each side and each stretc-h, respectively, by a number of additional oligonucleotides which, however, do not necessarily have to base-pair to each other.

RNAI has essentially the same design as siRNA, however, the molecules are significan tly longer compared to siRNFA. RNAi molecules typically comprise 50 or more nucleotides and I»ase pairs, respectively.

A further class of functional nucleic acids which are active based on the same mode of action as

SIRNA and RNA. is siNA. siNA is, e. g, described in international patent application

PCT/EP03/074654-. More particularly, siNA corresponcs to siRNA, whereby the siNA molecule does not comprise any ribonucleotides.

Antisense nucleic acids, as preferably used herein, are o ligonucleotides which hybridise based on base complementarity with a target RNA, preferabl y mRNA, thereby activating ZRNaseH.

RNaseH is activateed by both phosphodiester and phosphothioate-coupled DNA. Phosph_odiester- coupled DNA, however, is rapidly degraded by cellular nucleases with exception of phosphothioate-cotapled DNA. Antisense polynucleotidees are thus effective only as DNJA-RNA hybrid complexes. Preferred lengths of antisense nucleic acids range from 16 to 23 nucleotides.

Examples for this kind of antisense oligonucleotides aree described, among others, in US patent 5,849,902 and US patent 5,989,912.

py” » ! mpo06/0" 57 19

A further group of functional nucleic acids are ribozymes which are catalytically active nucleic acicys preferably consisting of RNA which basically comprise tvao moieties. The first moietsy sho~ws a catalytic activity, whereas the second moiety is responsitole for the specific interactior with the target nucleic acid. Upon in teraction between the target nucleic acid and the said moiets/ of —the ribozyme, typically by hybridisation and Watson-Crick= base-pairing of essentiallys complementary stretches of bases ora the two hybridising strands, t=he catalytically active moiety may~ become active which means that it cleaves, either intramolectalarly or intermolecularly, thes targeet nucleic acid in case the catalytic activity of the ribozyme is a phosphodiesterase activity_

Ribozymes, the use and design primciples are known to the one=s skilled in the art and, for exarmple, described in Doherty and Doudna (Annu. Ref. Biophys. “Biomolstruct. 2000; 30; 457- 75).

A still further group of fimctional rucleic acids are aptamers. Agptamers are D-nucleic acids whic=h are either single-stranded or double-stranded and which spec=ifically interact with a target mole=cule. The manufacture or selection of aptamers is, e. g., described in European patent EP 0 533 #38. In contrast to RNAI, siRNA, siNA, antisense-nucleotides and ribozymes, aptamers do not degrade any target mRNA but in®eract specifically with the seceondary and tertiary structure of a target compound such as a protein. Upon interaction with thes target, the target typically shows a change in its biological activi ty. The length of aptamers typi cally ranges from as little as to as much as 80 nucleotides, and pereferably ranges from about 20 to about 50 nucleotides.

Anotkaer group of functional nucleic acids are spiegelmers as, for example, described in international patent application WO ©98/08856. Spiegelmers are molecules similar to aptamers,

Howe=ver, spiegelmers consist either completely or mostly of L-—nucleotides rather than D- nucleotides in contrast to aptamers. Otherwise, particularly with re=gard to possible lengths of spiege=lmers, the same applies to spiegelmers as outlined in connectiomn with aptamers.

As m entioned previously, the presemt inventor has surprisingly found that the compound according to the present invention andl the respective compositions comprising such compound can bez particularly effective in transferring RNAi, and more particularly siRNA and siNA into a cell. It- is to be noted that although not wishing to be bound by any thheory, due to the particular mol peercentages of the helper lipid(s) contained in the lipid compositions according to the presen-t invention, which helper lipid can be either a PEG-free helper I=ipid or in particular a PEG- contairing helper lipid, surprising effects can be realised, more particularly if the content of any of this kind of heelper lipid is contained within th-e concentration range specified herein. In connection therewith, it is particularly noteworthy that if the composition according to the present invention contains a helper lipid comprising a PEG moiety, any delivers or transfection action using such PEG-derived helper lipid containi:ng composition is particul=nly effective in delivering nucleic acid, particularly RNAi molecules » most particularly siRNA, siNA, antisense nucleotides and rib>ozymes. The reason for this is that the present inventors haave surprisingly found that liposonmes containing more than about 4%, of PEG-containing helper lipid(s) are not active, whereas lipsosomes with less than 4% (preferrably less than 3%) do mediate functional delivery. Basically, the present inventors have discovered that the specific amoumt of PEG in the lipid compositions according to the present inventicon is suitable to provide for an effective transfection and delivery, respectively.

In a further aspect the present inventors have surprisingly found that the Lipicl compositions according to the pmesent invention which are prefer—ably present as lipoplexes or liposomes, preferably show an overall cationic charge and thus an excess of at least one positive charge.

More preferably, the lipid compositions exhibit a charge ratio negative : positive of from about 1: 1.3 to 1:5. Therefore, the present invention is this related in a further aspect to any lipid composition comprising at least one cationic lipid and a nucleic acid, preferably a RNAi, siRNA or siNA or any other of the functional nucleic acicls defined herein, having a charge ratio negative : positive o from about 1: 1.3 to 1:5. The castionic lipid is preferably any cationic lipid described herein. Th_e lipid composition comprises in a preferred embodiment any Telper lipid or helper lipid combiraation as described herein. Tn a preferred embodiment the composition according to the prezsent invention containing nucleic acid(s) forms lipoplexes. dn a preferred embodiment the terran lipoplexes as used herein refers to a composition composed of cationic lipid, neutral helper Jipid and nucleic acid.

The present inventors have also found that in particular -the molar ratio of siRNA amd the cationic lipid can be crucial for the successful application of ~ the lipid composition accwording to the present invention, especially in view of what has bee=n said above in relation to the cationic overall charge of the nucleic acid containing lipid form 1lations. Without wishing tos be bound by any theory it seems tknat] mole of cationic lipid, particu’ larly as disclosed herein, can provide for a maximum of thre ¢ positive charges per molecules, whereas the nucleic acid and more particularly the siRN_A molecules as disclosed herein, provide for a maximum off 40 negative charges per molecule. In order to reach an overall positive charge of the siRNA comtaining lipid formulations according to the present invention, the molar mratio can range from 0 to a Pmaximum of 0.075. A preferred mo Jar ration range is from about 0.02- to 0.05 and even more prefeerred is a molar ratio range of about 0.037.

Another surprising finding of the present inventors is that the composition accordimmg to the present invention exhibits particularly useful characteristics if the composition contains =a nucleic acid, preferably a siRNA. molecule or a siNA molecule, znd the charge ratio of nucIeic acid backbone phosphates to cationic lipid nitrogen atoms is abovat from 1:1,5 — 7, more prefe=rably 1 : 4. The term nucleic acid backbone phosphates as used herein refers to the phosphate momieties of the nucleic acid provided by the individual nucleotide forming such nucleic acid. T he term cationic lipid nitrogen atom as used herein refers to thosse nitrogen atoms provided. by the cationic lipid which preferably comprises a total of three positive charges. Said three positive charges are provided by twwo primary amino groups and the _guanidine group. For the pumpose of determining the charge provided by the nucleic acid backbone phosphates the fo-llowing assumptions are made: Each phosphate between two nucMeosides provides for one raegative charge and and 3’ terminal phosphate, if present, provides for two negative charges. “For the purpose of determining thes ratio of the charges provided by tlhe cationic lipid nitrogen atoms and the charges provided by the phosphate atoms it is assume=d that the charges are pre sent as described above although it has to be acknowledged that under the particular circum stances observed under in vitro and/or in vivo application the effective charge ratio might be different from the one specified above,

The above defined charge ratio provides for an efficient tramnsfer of the nucleic acid across a phospholipid bilayer membrane such as a cytoplasma membrane.

A further feature of the cormposition according to the presen-t invention which provides for its delivery characteristics, is its size distribution. PreferabBy, the size distribution of the composition according to the present invention being present as a dispersion is about 12-0 nm.

The size is preferably deterrmined by Quasi Elastic Light Scattering, as described in more detail in the example part.

The present inventors have surprisingly found that the compposition according to the pmresent invention is particularly suitable to deliver nucleic acids, prefer—ably functional nucleic acidss such as siRNA and siNA molecules, into cells. As outlined in —more detail in the examples, the compositions according to the present invention ares very active in delivering: said nucleic acids into the intracel lular space of endothelial cells, epithelial cells and cancer cells. There seems to be an even more increased specificity such that the delivery is particularly active in endothelial cells of vasculature, although other endothelial cells can also be infected usirg the composition according to thes present invention. A particularly effective transfection occumrs with endothelial cells of vasculature, more specifically vasculature which is the result of meoangiogenesis as induced by tumeors. Other vasculature which might be addressed is the vasculature of kidney, heart, lung, liver and pancreas.

It is to be ackmowledged that the composition according to the present invention is also beneficial insofar as it is particularly mild or non-toxic. Such lack of teoxicity is clearly advantageous over the compositions of the prior at as it will significantly contribute to the medicinal benefit of any treatment using this kind of composition by avoiding side efects, thus increasing patient compliance and particular feorms of administration such as bolus administration. T he latter is, as may be taken from thme example part herein, evident from animal studies.

It is within the pmresent invention that the compositiomn and more particularly thme pharmaceutical composition may comprise one or more of the aforementioned biologically a ctive compounds which may be cortained in a composition according teo the present invention as —pharmaceutically active compound and as further constituent, respecti-wely. It will be acknowleciged by the ones skilled in the art —that any of these compounds can, im principle, be used as a ZDharmaceutically active compound. Such pharmaceutically active compound is typically directecd against a target molecule which is involved in the pathomechanism of a disease. Due to thes general design principle and modle of action underlying the various beiologically active compoumnds and thus the pharmaceutically active compounds as used in cormection with any aspect of the present

Invention, virtuall-y any target can be addressed. Accordingly, the compound zaccording to the present invention and the respective compositions containing the same can be used for the treatment or preve=ntion of any disease or diseased cordition which can be addreessed, prevented and/or treated usirag this kind of biologically active ccompounds. It is to be acl <mowledged that apart from these biologically active compounds also ary other biologically activee compound can be part of a composition according to any embodiment of the present invention. Preferably such other biologically active compound comprises at least one negative charge, preferably under conditions where ssuch other biologically active compound is interacting or commplexed with the compound according to the present inventiorn, more preferably the comp-ound according to the present invention which is present as a cationi ¢ lipid. .

As used herein, a biologically active cormpound is preferably any compound which is biologically active, preferably exhibits any iological, chemical and/or physical effects on a biological system. Such biological system Es preferably any biochemic al reaction, any cell, preferably any animal cell, more preferabl y any vertebrate cell and most preferably any mammalian cell, including, but not limited to, any human cell, any tissuee, any organ and any organism. Any such organism is preferably selected from the group comprising mice, rats, guinea pigs, rabbits, cats, dogs, monkeys and Imumans.

It is also withira the present invention that amy of the compositions according to the present invention, more particularly any pharmaceutical composition according to the present invention may comprise ary further pharmaceutically active compound(s).

The composition_, particularly the pharmaceutical composition according to the present invention can be used for various forms of administrastion, whereby local adminiswration and systemic administration are particularly preferred. Even more preferred is a route of administration which is selected from the group comprising intramaascular, percutaneous, subcutaneous, intravenous and pulmonary amdministration. As preferably used herein, local administration means that the respective compcosition is administered in closes spatial relationship to the cell, tissue and organ, respectively, to which the composition and the biologically active compoun-d, respectively, is to be administered. As used herein, systemic aciministration means an adm inistration which is different from a l~ocal administration and more “preferably is the administrati on into a body fluid such as blood andl liquor, respectively, whereby the body liquid transports thes composition to the cell, tissue and eorgan, respectively, to which the composition and the biologically active compound, respec=tively, is to be delivered.

As used herein, thme cell across the cell membrare of which a biologically acstive compound is to be transferred by means of the compound and composition according to thes present invention, respectively, is preferably an eukaryotic cell, nore preferably a vertebrate acell and even more preferably a mamrmalian cell. Most preferably thee cell is a human cell.

Any medicament which can be manufactured using the compound and ¢ omposition according to the present invention, respectively, is for the treatment and prevention of a patient. Preferably such patient is a vertebrate, more preferably a amammal and even more Preferably such mammal is selected From the group comprising mice, rats, dogs, cats, guinea pigzs, rabbits, monkeys and humans. In a further aspect the compound ancl composition according #to the present invention can be used as a transferring agent, more preferably as a transfection agert.

As preferab 1y used herein a transferring agemt is any agent which iss suitable to transfer a compound, mmore preferably a biologically acti ve compound such as a goharmaceutically active compound a cross a membrane, preferably a cell membrane and more oreferably transfer such compound irato a cell as previously described herein. Preferably, the cel Is are endothelial cells, more preferably endothelial cells of vertebrates and most preferred endotinelial cells of mammals such as mice, rats, guinea pigs, dogs, cats, monk-eys and human beings.

In a still futher aspect the present invention is related to a method for transferring, more particularly taransfecting, a cell with a biologicakly active compound. In a first step, whereby the sequence of Steps is not necessarily limited, the cell and the membrane ard cell, respectively, is provided. In z= second step, a compound accordirg to the present inventiorm is provided as well as a biologically~ active compound such as a pharmaceutically active compo und. This reaction can be contacted with the cell and the membrame, respectively, and dime to the biophysical characteristics of the compound and the composition according to the —present invention, the biologically active compound will be transferred from one side of the membrane to the other one, or in case the membrane forms a cell, from outside the cell to within the cell. It is within the present inveration that prior to contacting the cell and the membramne, respectively, the biologically a ctive compound and the compourad according to the present invention, i. e. the cationic lipid, are contacted, whereupon preferably a complex is formed and such complex is contacted withm the cell and the membrane, respectively.

In a further aspect of the present invention thes method for transferring a biologically active compound aned a pharmaceutically active comapound, respectively, cormprises the steps of providing the cell and the membrane, respectively, providing a composi-tion according to the present invention and contacting both the composition and the cell and the membrane, respectively. Int is within the present invention hat the composition masy/ be formed prior or during the contacting with the cell and the membr.ane, respectively.

In an embodiment of any method for transferring a biologically active compsound as disclosed herein, the methosd may comprise further steps, preferably the step of dete cting whether the biologically actives compound has been transferrecl. Such detection reaction strongly depends on the kind of biolo gically active compounds transferred according to the me=thod and will be readily obvious fo-r the ones skilled in the art. Tt is within the present invention _ that such method is performed on amy cell, tissue, organ and organisc as described herein.

The present inventtion is further illustrated by reference to the following figu-res and examples from which further features, embodiments and advantages of the present invent=ion may be taken.

More particularly,

Fig. 1 shovavs the basic design of the cationic lipid according to the prese=nt invention;

Fig. 2 shows the synthesis of N-oleyl-palmiitylamine which is a possible= starting material for tThe synthesis of the compounds according to the present invention, whereby such synthesis is the one according to the prior art as described in US 6,395,713;

Fig. 3 depicts the synthesis of N-oleyl-paJmitylamine which is an inmportant starting matexrial according to the present inve=ntion;

Figs. 4-9 depict the synthesis of B-arginyl-2,3-amino propionic acid-N-p=almityl-N-oleyl- amide= trihyrdochloride, B-arginyl -2,3-diamino propionic &acid-N-lauryl-N- myrisstyl-amide trihydrochloride and e-arginyl-lysine-N-lauryl-N- -myristyl-amide trihycdrochloride;

Fig. 10 depicsts the synthesis of an alternate «cationic head group which Ms an alternative compeonent for the synthesis of the cationic lipids according to the present inven@tion;

Fig. 11 depict=s an alternative synthetic rowmte for the synthesis of b-eta-arginyl-2,3- diamimopropionic acid-N-palmityl-N-oleyl-amide trihydrochlorides.

Figs. 12A and 12B depict the size distribution of lipid formulations a-ccording to the present invention and the impact of extrusion and high-preessure homogenisation, respectively;

Fig. 13 depicts the result of a Westen Blot analysis of an ERNAi containing lipid formulation being exposed to cryoprotectants and stored at different temperatures;

Fig. 14 depicts the result of a Western Blot analysis on the impaact of different siRNA loads on lipid formulations differing in their helper lipid;

Fig. 15 depicts the result of a Western Blot analysis and thes impact of different concentrations of PEG-substituted lipids;

Fig. 16 depicts the experimental set up used to generate a Ras=V12-dependent tumor mouse model and its use in testing various formulations;

Figs. 17A, 17B and 17C depict diagrams indicating the tumor volumes as a function of days post cell challenge using different formulatiomns;

Fig. 18a depicts the result of a Western blot analysis of the effect of= naked and lipoplexed

PKN3 specific siRNA;

Fig. 18b are confocal microscopyphotographs showing the intrace=liular distribution of naked and formulated siRNAs;

Fig. 18¢ are epifluoresecence microscopy (upper panels) and - confocal microscopy photographs (lower panel) depicting the distribution of lipo somal formulated and naked siRNAs in liver;

Fig. 18d are epifluorescence and confocal microscopy photographs of endothelial cells targeted with liposomal formulated siRNAs;

Fig. 18¢e are fluorescence microscopy photographs of endothelial cellss of different tumors;

Fig. 19a is a schematic illustration of the mode of action of PTEN directed SIRNA on DNA synthesis, and shows the result of a Western Blot analysis using different siRNA species and immunofluorescence microscopy photographs omf HELA cells treated with said different siRNA species;

Fig. 19b depicts pictures of endothelial cells treated with different siFRNA molecules and a diagram representing the result of a BrdU assay in liver endosthelial cells

Fig. 19¢ depicts pwictures of endothelial cells treated with different siRNA mole=cules and 2 diagram -representing the result of a BrdU assay in tumor endothelial ce=lis;

Fig. 20a depicts the result of a Western blot analysis for determining potzent siRNA molecule=s for efficacious CD31 knock-do-wn;

Fig. 20b is a diagram illustrating the effect of anti-«CD31 siRNA on CD 31 mRNA levels in different - organs of mice;

Fig. 20c shows thee result of a Western blot analysis for determining the efficacy of CD31 protein l=mock-down in different organs of mice using anti CD®31 siRNA moleculess;

Fig. 20d shows tlie result of in vivo knock-edown of CD 31 protein by direct immunos®aining of paraffin tumor secti-ons of mice treated with a nti-CD 31 siRNA meolecules;

Fig. 21a depicts thme result of a Western blot analwwsis studying the efficacy of anti-CD31 siRNA ard anti-PTEN siRNA molecules on CD31, CD34, PTEN and p-Akt knock-dowwn;

Fig. 21b . is a diagraam illustrating the effect of different patterns of lipoplex adnministration on body w=eight of test animals as a functio-n of time;

Fig. 21c represents diagrams illustrating the effect of different anti-CD31 siRNA _ treatment regimens on the volume of two different tumor xenografts; and

Fig. 21d is 2 diagram illustrating the inhibition of growth of established PC-3 =xenografts under an a—nti-CD31 treatment regimen.

Example 1: Synthesis of N-oleyl-palmityl amine acco rding to the prior art

N-oleyl-palmityl amine Fs an important starting materia for the compounds accordi_ng to the present invention. The N--oleyl-palmityl amine can, in principle, be synthesised as desscribed in

US 6,395,713. The respective reaction scheme is depicted in Fig. 2. However, thee starting material is oleyl amine of technical grade as provided by, «. g, Fluka. An analysis of thHs starting material by gas chromato geraphy shows a purity of = 70 %w, whereby 30 % of the materi al consist of amine having different. chain lengths. The reason for thmis could be that the material =as such is obtained from plant ssources. Combining both oleeyl amine and 1-bromohe=xadecane (palmitylbromid) yields M-oleyl-palmityl amine after reacting both starting materials at 100 to 120° C for 30 minutes. Thue yield is about 83 %.

Example2: Synthesis of N-palmityl-oBeyl amine according to the present invention

A_ new synthesis has been perceived by the present inventor in connection with the compournds according to the present invention (Fig. 3). This new reaction scheme is based on the finding: of thee present inventor that the high amount of impurities is affecting the quality of the transferring agent prepared based on this starting matesrial. Accordingly, the reaction starts using oleic acid having a purity of > 99 % as shown by gas chromatography and conta=cting such oleic acid with ethylchloroformate, TEA and CH,Cl, and reacting the thus obtained mixed carboxylic-carboric anhydride with hexadecylamine (palmitylaamine) having again a purity— of > 99 % as shown by gas chromatography. The reaction product N-palmityl-oleoyl amide [#M ] is subsequently react-ed with LiAlH4 (in THF) resulting in 85 % N-palmityl-oleyl amine [#22] which is present as a colourless crystalline solid.

The more detailed reaction conditions are omutlined in the following.

Syxathesis of N-palmityl-oleoyl amide #17] 2.62 ml (27.5 mmol) chloroformic acidl ethyl ester are dissolvec in 30 m] anhydroums dichloromethane in a 250 ml nitrogen flassk according to Schlenk uneder argon inert gas an d cooled to 0° C. A solution of 7.93 ml (25 mmol) oleic acid and 4.16 ml «30 mmol) triethylamin_e in 4 0 ml anhydrous dichloromethane are adcIed dropwise under steering wwithin 20 minutes. Afie=r steering on the ice bath for 30 minutes a sol ution of 6.64 2 (27.5 mmol) palmitylamine in 50 mm]

CHC; is rapidly added dropwise and the rmixture is steered at room temperature for 2 hourss.

Subsequently, the solution is washed three times with 40 ml water each, the organic phase dried over Na;SO, and the solvent removed using a rotary evaporator. The reesidue is re-crystallisecl froma 100 ml acetone. 11.25 g (22.3 mmol) cemresponding to a yield of 89 % of a colourless solic is obtained.

Synthesis of N-palmityl-oleylamine [#2] m1 IM LiAlIH, in ether are provided under argon inert gas in a 250 ml wthree-neck flask having— a dropping funnel and a reflux condenser znd subsequently a solution of 7.59 g (15 mmol) palmityloleoylamide in 80 ml THF added dreopwise within 20 minutes. T he mixture is refluxed for 2.5 hours, €hen another 5 ml 1 M LiAlH, in ether is added and refluxed for another 2.5 hours.

Excess hydrides is decomposed using 6 M NaOH under ice bath cooling armd the precipitate is filtered off. Thhe precipitate is extracted twice with 40 ml of hot MtBE esach, the combined organic phasess dried over N2;SO; and the soMvent removed using a rotary evaporator. The residue is crystallised from 100 ml MtBE at -20° C. 6.23 g (12.7 mmol) corre=sponding to a yield of 85 % of a camlourless crystalline solid are obtaimed.

Example 3: Synthesis of Boc-Dap(Fmoc)-N-pelamityl-N-oleyl-amide [#3] 521 mg (1.06 mmol) N-oleyl-palmitylamine in 1 0 ml anhydrous dichloromethane are dissolved in a2 50 ml roun d-bottom flask and 289 mg (1.17 mmmol) EEDQ are added. Subosequently, 500 mg (1.17 mmol) Beoc-Dap(Fmoc)-OH are added under steering and the mixture is steered at room temperature foam 20 hours. The solution is tramsferred with 80 ml dichloromethane into a separating funmel and washed three times with 20 ml 0.1 N HCI each and. once with 20 ml saturated NaHCO; solution. After drying over ™Na;SO, the solvent is remowed using a rotary evaporator (Fig. 4). A yellowish viscous oil is obtained which is not further pumrified. In thin layer chromatography~ using hexane/ethylacetate of 1:1 = R¢ 0f 0.70 was observed.

Example 4: _S ynthesis of Boc-Dap-N-palmityl—N-oleyl-amide [#4] 1 g Boc-Dap(Frmoc)-N-palmityl-N-oleyl-amide ramw product were dissolved isn 8 ml anhydrous dichloromethane= in a 50 ml round-bottom flask. 3 ml diethylamine were adcaled and steered at room temperatux=e (Fig. 4). Thin layer chromatography control of the reaction showed that after 4.5 hours the resaction of the starting product was completed. The volatile =components were removed by a rotary evaporator and the residue is chromatography purified using 40 g silica gel 60 (Merck) usimg hexane/ethylacetate 5:1. The product was eluted using= a step gradient consisting of eth_ylacetate, ethylacetate/methanol 4 =1 and dichloromethane/mettaanol 4:1. 576 mg (0.85 mmol) Bocs-Dap-N-palmityl-N-oleyl-amide were obtained as a yellow visecous oil.

Example 5: Synthesis of tetra-Boc-[B-arginy®-2.3-diaminopropionic _aciid-N-palmityl-N- oleyl-amide] [#5] 576 mg (0.85 mmol) Boc-Dap-N-palmityl-N-oley~l-amide were dissolved in ~10 ml anhydrous dichloromethane in a 100 ml round-bottom flask sand 210 mg (0.85 mmol) EEEDQ and 403 mg

(0.85 mmol) Boc-Arg(Boc),-OH were added under steering (Fig. 5). The mixture was steered under argo atmosphere at room temperature for 240 hours. Subsequently, the dichloromethane is removed by a rotary evaporator and the residue im 100 m! MtBE transferred into a separating funnel. Thes organic phase was thoroughly washeed with 0.1 N HCl, 1 N NaOH and saturated

NaHCO; solution, dried over Na;SO, and the solv-ent removed by a rotary eva porator. The raw product was subsequently purified by flash chrormatography (Combiflash Retrieve; Isco Inc.) using hexame/ethylacetate as eluent. 694 mg (0.61 mmol) corresponding to a yE.eld of 72 % of a colourless viscous oil was obtained.

Example 6= Synthesis of B-arginvl-2,3-diamin opropionic _acid-N-palmity-1-N-oleyl-amide trihydrochloride [#6] 694 mg (0.461 mmol) well dried tetra-Boc-[B-argirayl-2,3-diaminopropionic aci d-N-palmityl-N- oleyl-amide ] were provided under argon atmosphere in a 25 ml nitrogen flask according to

Schlenk andl 8 ml 4N HCI in dioxane added (Fig. 5°). The mixture was steered under argon inert gas at room temperature for 24 hours, whereby product precipitated as amorphous and partially as wax-like solid from the solution after about 6 t< 8 hours. After completion of the reaction (thin layer ceontrol using CHCly/MeOH/NH,OH 65:225:4) any volatile component—s were removed under high vacuum. 489 mg (0.58 mmol) B-argirayl-2,3-diaminopropionic aci_d-N-pamityl-N- oleyl-amide were obtained as trihydrochloride.

Example 7: __Svnthesis of N-lauryl-myristyl amin _e [#7] 18.54 g (1000 mmol) dodecylamine (laurylamine)., 6.36 g (60 mmol) Na,CC€D; and 50 mg tetrabutyl ammonium iodide (TBAT) were suspendecd in 100 ml anhydrous DMF" in a 500 ml 3- neck flask having a reflux condenser and a dropping funnel. A solution of 16.4 nl (60 mmol) 1- bromo tetradescane in 100 ml anhydrous dioxane were added dropwise at 100° C Over a period of 110 minutes and the mixture was steered for anothemr 3.5 hours at 100° C (Fig. &). The solution was filtered at a temperature as hot as possible. The crystalline solid which precipitated at 4° C over night, w-as removed and was washed with a littl e of cold methanol. Subsequently, the solid was recrystallised from 200 ml methanol. 9 g of colourless leaf-like crystals were obtained which are re—crystallised from 100 ml MBE. The crystals which precipitated at 18° C, were sucked off from a cooled frit and washed with colcd MtBE. 7.94 g (21 mmol) eof a colourless crystalline solid were obtained, corresponding to a yield of 35 %.

Example 8: Synthesis of Boc-Dap(Frmoc)-N-lauryl-N-myristyl amide [#8] 715 mg (1.68 mmol) Boc-Dap(Fmoc)-OH were dissolved in 15 ml anhydrous dichloromethane in a 50 ml round-bottom flask and 420 mg (1.7 mmol) EEDQ were adlded. The mixture was steered. at room temperature for 45 minutes and subsequently a solution of 641 mg (1.68 mmol)

N-laurs/l-myristyl amine in 25 ml anhydrous dichloromethane was slowly added dropwise within 60 mirmautes (Fig. 6). After a reaction time of 20 hours the solvent was removed by a rotary evaporator and the residue transferred with 100 ml MtBE into a separatimeg funnel. The solution was thoroughly washed with 0.1 N HCI sand saturated NaHCO; solution, the organic phase dried over N.a,SO, and the solvent removed by a rotary evaporator. 1.02 g of a raw product were obtained which was purified by flash chromatography (Combiflash Retrieve; Isco Inc.) using hexane/ethylacetate as eluent. 607 mg pure product were obtained as colouxless, very viscous oil.

Thin laser chromatography using hexane /ethylacetate 1:1 provided a R¢ of 0.58.

Example9: Synthesis of Boc-Dap-N-B auryl-N-myristyl amide [#9] 607 mg Boc-Dap(Fmoc)-N-lauryl-N-m yristyl amide were dissolved in 8 ml anhydrous dichlorosmethane in a 50 ml round-bottorm flask (Fig. 6). 3 ml diethylamime were added and the reaction steered at room temperature for 43.5 hours. The volatile constituents were removed using a rotary’ evaporator and the residue was purified by chromatography usimg 40 g silica gel 60 (Merck) with hexane/ethylacetate 5:1. Thae product was eluted by a step gradient consisting of ethylace tate, dichloromethane and dichlosromethane/methanol 3:1. 372 mg (0.655 mmol) Boc-

Dap-N-1auryl-N-myristyl amide were obtained as yellowish, viscous oil.

Example 10: Synthesis of _tetra-Boc—|B-argin yl-2.3-diaminopropioni ¢ _acid-N-lauryl-N- myristyl amide] [#10] 372 mg (0.655 mmol) Boc-Dap-N-lauryl—N-myristyl amide were dissolved in 8 ml anhydrous dichlorormethane in a 50 ml round-bottora flask and 162 mg (0.655 mmol) EEDQ and 311 mg (0.655 mumol) Boc-Arg-(Boc),-OH were a dded under steering (Fig. 7). The mixture was steered at room temperature for 20 hours. Subsesquently, the dichloromethane wras removed using a rotary evaporator and the residue was transferred with 80 ml MtBE into a separating funnel. The organic phase was thoroughly washed wwith 0.1 N HCl, 1 N NaOH and saturated NaHCO,

solution, dried over Na,SOs and the solvent removed by a rotary evaporator. The raw product was subsequently purified by= flash chromatography (CombifSlash Retrieve; Isco Inc.) using a step gradient of hexane/ethylacet=ate. 500 mg (0.5 mmol) of a colourless viscous oil weere obtained, corresponding to a yield of 765 %.

Example 11: Synthesis of [3-argin y}-2.3-diaminopropioniic acid-N-lauryl-N-mymristyl amide trihydrochlor—ide [#11] 511 mg (0.5 mmol) well dred tetra-Boc-[B-arginyl-2,3-«diaminopropionic acid—N-laury]-N- myristyl amide] were provided under argon in a 25 ml argo flask according to Schmlenk and 10 ml 4 N HCl in dioxane were= added (Fig. 7). The mixture was steered under argon inert gas at room temperature for 24 hovmrs, whereby product precipitated as partially amorpho-us, partially wax-like solid from the solut=ion after 6 to 8 hours. Upon completion of the reactior (thin layer chromatography control usimmg CHCly/MeOH/NH,OH 65:255:4) all volatile components were removed under high vacuum. 323 mg (0.5 mmol) B-arginyl-2_,3-diaminopropionic ac=id-N-lauryl-

N-myristyl amide in the form of the tri-hydrochloride were obtained.

Example 12: Synthesis of Beoc-Lys(Fmoc)-N-lauryl-N-myr—istyl amide [#12] 937 mg (2 mmol) Boc-Lys(Frmoc)-OH were dissolved in 10 rl anhydrous dichloromethane in a =50 ml round-bottom flask andl 495 mg (2 mmol) EEDQ weree added (Fig. 8). The mixture was ssteered at room temperature for 60 minutes and subsequently a solution of 764 mg (2 mmol) N-

Baurly-myristyl amine in 30 rl anhydrous dichloromethane was slowly added in -a dropwise manner within 120 minutes. Amfler a reaction time of 20 hourss the solvent was remowed using a rotary evaporator and the resiadue transferred with 100 m! M_tBE into a separating Tunnel. The

SSolution was thoroughly washe=d with 0.1 N HCI and saturated® NaHCO;, the organic phase dried

Over Na;SO, and the solvent reemoved using a rotary evaporat-or. 1.757 g of a raw preoduct were

Obtained which was purified ussing flash chromatography with. hexane/ethylacetate 4: MW as eluent. 1..377 g pure product is obtai-ned as colourless, very viscouss oil. Thin layer chromatography umsing hexane/ethylacetate 1:1 geave a Reof 0.57.

Example 13: Synthesis of Boc-Lys-N-laurvi-N-myristys1 amide [#13] 1377 g Boc-Lys(Frmoc)-N-lauryl-N-myristyl-amide were dissolved in 16 mul anhydrous dichloromethane in 2 50 ml round-bottom flask. 6 ml die=thylamine were added anc the mixture was steered at room atemperature for 5 hours (Fig. 8). T he volatile components w—ere removed using a rotary evaporator and the residue was purified by chromatography using 4) g silica gel 60 (Merck) with hexame/ethylacetate 5:1. The product wass eluted using a step gradie=nt consisting of ethylacetate, dichloromethane and dichloromethane/me-thanol 3:1. 556 mg (0.911 mmol) Boc-

Lys-N-lauryl-N-myrisuatyl amide were obtained as yellowish viscous oil as well as 119 mg of a mixed fraction.

Example 14: Synthesis of tetra-Boc-[e-arginyl-lysine-N -lauryl-N-myristyl amide] [#14] 556 mg (0.911 mmol) Boc-Lys-N-lauryl-N-myristyl-amid_e were dissolved in 40 m 1 anhydrous dichloromethane and 2226 mg (0.911 mmol) EEDQ and 4.33 mg (0.911 mmol) Boc—Arg(Boc);-

OH were added under steering (Fig. 9). The mixture wass steered at room temperature for 20 hours. Subsequently, thme dichloromethane was removed using a rotary evaporator and the residue was transferred with 8«0 ml MBE into a separating funne=1. The organic phase was thoroughly washed with 0.1 N HCCI and saturated NaHCO; solution, dried over Na,SO; and the solvent removed using a rotary evaporator. The raw product -was subsequently purifie d by flash chromatography (Comboiflash Retrieve; Isco Inc.) using a hexane/ethylacetate step gradient. A colourless, viscous oil ~was obtained with a yield of 730 omg (0.684 mmol) corresporading to 75 %.

Example 15: Synthesis of g-arginyl-lysine-N-lauryl-N—myristyl amide trihyd rochloride [#15] 730 mg (0.684 mmol) well dried tetra-Boc-[e-arginyl-lys—in-N-laurly-N-myristyl amide] were provided under argon in a 25 ml argon flask according to Schlenk and 10 m1 4 N HC! in dioxane were added (Fig. 9). Thee mixture was steered under argon Mnert gas at room temperasture for 24 hours, whereupon produact precipitated from the solution zs an amorphous, partiallmy wax-like solid after about 8 houcrs. Upon completion of the reaction such as controlled by thin layer chromatography using CCHCly/MeOH/NH,OH 65:25:4, all volatile components weres removed

2008 70nd unde>r high vacuum. 491 mg (0.6533 mmol) &- arginyl-lysin-N-laurly-N-myristyl amide were obtamned as tihydrochloride.

Exarwple 16: Synthesis of Tri-Basc-y-carbamidino-o.y-diamin © butyric acid [#16] 1.31 g (6 mmol) Boc-Dab-OH were provided in 15 ml acetoni tile in a 100 ml round-botiom flask and 12 mmol diisopropyleth! amine (DIPEA) were added (Fig. 10). Subsequently water was added dropwise until a part of -the Boc-Dab-OH dissolved an d subsequently 1.96 g (5 mmmol) 1,3-d 3-Boc-2-(triflucromethylsulforyl) guanidine were added. Th e mixture was steered at mroom tempeenature for 12 hours, whereupon the acetonitrile was removed using a rotary evaporator_. The aqueus residue was diluted with 5 ml water and 50 ml] diclmloromethane were added. The reaction is acidified to a pH 2 by adlding 2 N HCI under steering amd subsequent separation ofthe orgammic phase. The aqueous phase ~was extracted with 50 mi dich loromethane and the combmined organ ic phases were subsequently veashed with some of diluted H<Cl and saturated NaCl solu_tion.

The corganic phase was dried over Na,SO4 and the solvent was removed using a rotary evapostator. The residue was purified using chromatograplhy on silica gel 60 umsing } hexan: e/ethylacetate 2:1. 1.138 g (2- 47 mmol), corresponding to a. yield of 50 %, of a coloumrless amorphous solid was obtained.

Exam ple 17: Synthesis of beta -arginyl-2.3-diaminopropiomic acid-N-palmityl-N-ol_eyl- amide trihydrochlor-ide [# 6] 1.225 _g (6 mmole) Boc-Dap-OH in 15 ml absolute CH2CI2 are smispended in a 250 ml Schlenk flask czomprising a dropping funnel winder an argon atmosphere and 1.72 ml thrimethylamine are added. A solution of 1.52 ml (12 mxmole) TMSCI in 30 ml absohmte CH2CI2 is added dropwise within 15 to 20 minutes at room termperature under vigorous stirring. In the meantime 941 mg (5.8 mmole) carbonyl diimidazole iss dissolved in 8 ml absolute €CH2CI2 in a 100 ml Schies=nk flask under argon atmosphere. A so lution of 2.66 g (5.6 mmole) Boc-Arg(Boc)2-OH in 25 ml absolute CH2CI2 is added dropwise within 15 to 20 minutes at room temperature and un der stirring. Both reaction solutions are stirred at roomp temperature for 4 h. Subsequently, 832 ul ( 6 mmo Te) triethyl amine are added to the first solution and the secomd solution is added dropwise within 15 to 20 minutes through the dropping funnel at room temperature under argzon atmosp_here. After 15 to 20 minutes 30 ml! water are added, vigorously stirred for 45 minutes a=and thee solution is adjusted to a pH of 2. The organic phase is separated and the aqueous phase extracted several times with CH2CI2. The combined organic plaases are dried with a saturated soslution of NaCl and sodium sulfate and the solvent removed using a rotary evaporator. The glass-like residue is purified using flash chromatography on silicagel using dichloromethane as elwient. 2.74 g (4.15 mmole; 7=4%) of a colourless, amorphous soliad is obtained [compound 17].

Thais solid is reacted with oleyl pelmityl amine [#2] under conditions wevhich are essentially analogous to thes one of Example 10, whereb=y the temperature is set to 35 to 40 °C (yield 72 %). The intended final product B -arginyl-2,3-diaminopropionic acid-N-palmityl-N-oleyl—amide trihydrochloride [#6] is ob®ained upon cleaving off the Boc protection groups as described ira Example 11. The thus obtained product can be further purified umsing flash chromatography on RP- 18 silica gel using MeOH/ water as elu ent.

Ex=ample 18: Manufacture eof complexes consisting of cati onic liposomes and siRNA (Lipoplexes)

Lip oplexes consisting of catieonic liposomes and siRNA were manufactured using standard teckanologies known in the art such as lipid film/cake rehydratiora, ethanol injection procedure, revesrsed phase evaporation or detergent dialysis procedure [c.f. Liposomes as Tools in Basic

Res earch and Industry; Jean 2. Philippot and Francis Schuber; CCRC Press January 1995 und

Liposome Technology: Prepar=ation of Liposomes:001 Gregory (Gregoriadis CRC Press I Lic.

April 1984).

The thus obtained liposomes which are also referred to herein as lipoplexes in combination with nucl eic acids such as siRNA comprise as the lipid B-arginyl-2 3-diaminopropionic acid-N- palmityl-N-oleyl-amide trihydreochloride and additionally either 1,2-diphytanoyl-sn-glycero-3- phossphoethanolamine or 1,2-dioleyl-sn-glycero-3-phosphoethanolammine, whereby the use of 1,2- diph_ytanoyl-sn-glycero-3-phosp~hoethanolamine is preferred. The lipid fraction of such liposomes and lipoplexes, respectively, was 50 mol% beta-arginyl-2,3-diaminopropionic acid-N- palmuityl-N-oleyl-amide trihydr=ochloride and either 50 mol% L ,2-diphytanoyl-sn-glycero-3- phos-phoethanolamine or 50 mol % 1,2-dioleyl-sn-glycero-3-phosphoethanolamine.

The combination of 50 mol% B-arginyl-22 3-diaminopropionic acid-N-palmityl-N-oleyl-amicie trihydrochloride and 50 mol% 1,2-dipkhytanoyl-sn-glycero-3-phospBhoethanolamine is alsso referred to hexein as atuFect.

It is to be unclerstood that in principle any other lipid and lipid compossition as disclosed herefin can be manufzactured using the previously mentioned techniques as well as the further processinmg steps.

The liposomes and lipoplexes, respectively, are subjected to further processing steps so as to trirm them with regzard to size, polydispersibility &and lamellarity. These charac=teristics can be adjusted by sonication, extrusion such as through =orous membranes, and homogenisation, preferabl=y high pressure Fnomogenisation.

The thus formed liposomes or lipoplexes wesre characterised by photon correlation spectroscope’ with Beckman —Coulter N § submicron particle analyser and the results o-f such liposomes either sized by extrusion or by high-pressure ho-mogenisation are depicted —in Fig. 12A and 12B_, respectively.

From Fig. 12A it can be taken that the size clistribution of the liposomes can be modified using- different memb»ranes having different size exclusions, in the present case 1,000 nm and 400 nm, respectively. In. both cases, the extrusion stepo was repeated 21 times. It is, however, within the present invention that the size exclusion can be from about 50 to 5000 nm_, and that the extrusion steps can be repeated 10 to 50 times.

As may be takem from Fig. 12B high-pressure homogenisation is also a sui” table means to modify the size distribution of the liposomes, whereby upon applying such high-pressure homogenisation the size of the liposomes depends on the number of hommnogenisation cycles to which the liposomes were subjected. Typical pressure ranges are from 100-2500bar, whereby in the present case the applied pressure was 1,500) bar.

Example 19: Sworage stability of atuFect

If the compositions disclosed herein are typi_cally used as pharmaceutical compositions, it is essential that such pharmaceutical formulationss are stable to storage conditions. In order to study the storage stability an siRNA was designed against tumor suppressor PTEN which was forrmulated using atuFect as described ix example 18.

Moe particularly, liposomes were manufactured using a lipid stock sol ution with the final stock conscentrations being recited below, by lipid film rehydration in 300 mM sucrose solution, followed by extrusion and high pressure homogenisation, respectively. The thus obtained liposomes were mixed with the siRMNIA molecules described below at a mol ratio of 1:1; alternatively the lipid layer could be rehydrated in the presence of siRINJA and the thus obtained lipogplexes extrudated of homogenized.

The siRNA molecules were the followin g antisense PTENAV 10: 5’ uaaguucuagcuguggugg-P 3°; senses PTENBV10 5’ ccaccacagcuagaacuua-P 3°; swhemreby bold nucleotides indicate that the respective nucleotoide is 2°-O®-methyl.

The 1 dpoplexes were incubated on HeLa cells in the presence of serum co-ntaining medium for 48 h at different concentrations (nM of siR NA molecule is shown in figur-e 13). The immunoblot with whole cell extracts using a pl10a (loading control) and PTEN specific antibodies was perfomrmed as described previously (Stand ard-Wester-Protocol).

Suitable cryoprotectants include, howewer, are not limited to, sucrose, trehalose, maltose, cellobwiose, raffinose, galactose, mannitole and PEG. In the present example, a 300 mM sucrose soluticon was used as a carrier for the atuF ect formulation containing the FP TEN targeting siRNA.

The final stock concentrations were total lipids 1,445mg/ml and 15 pu M PTEN-siRNA. The solution was kept either at room temperature, stored at 4° C for seven damys or stored at —80° C for se~wen days. Said solution was diluted in serum-containing medium to the indicated final conceratration (20, 10, 5 nM). Tests were performed on HeLa cells with a_ cell density of 40,000 well. “The results are depicted in Fig. 13 from which it can be taken that freezing atuFect contairaing siRNA in a cryoprotectant ancl storing the same at —-80° C for seven days is, after thawin_g, as effective as if it was kept at 4° C.

Example 20: Lipid composition and siRENA load

Two di ferent types of lipid formulations were prepared. Lipid formula=tion 01 consisted of 50 mol% B-arginyl-2,3-diamino propionic acid-N-palmityl-N-oleyl-amicde trihydrochloride as cationic lipid, and of 50 mol% 1,2-diphytanoyl-sn-glycero-3-phosphos ethanolamine, whereas lipid formulation 02 consisted of 50 mol%g B-arginyl-2,3-diamino propiconic acid-N-palmityl-N- oleyl-armide trihydrochloride as cationic lipid, and of 50 mol% 1.,2-dioleyl-sn-glycero-3- phosphOethanolamine. Each lipid formulati on contained an siRNA directe=d against PTEN, (stock concentmation was 15uM siRNA and 1,445mg/ml lipids ), whereby the molarity of the siRNA was titrated on the cells leading to an end concentration of 1 uM, 500 nMw/, 100 iM and 50 nM, respectiwely.

These P"TEN specific RNAi containing lipid formulations were administered to a mouse cell line (B16V, ATCCNo.:. CRL6475) grown uncler standard cell culture comditions in Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to commtain 1.5 g/L sodium bicarbon ate and 4.5 g/L. glucose, 90%; fetal bovine serum, 10%. The cel densitly was 40.000 cells / 6 well and after 48 hours the cells were lysed and subjected to a We=stern Blot analysis the result of “which is depicted in Fig. 14. The signal obtained with an monocI: onal antibody specific for the k-inase PRK2 (Becton Dickinson) wwas used as a loading control in comparison to the

PTEN signal (monoclonal antibody, Santa Cxuze, CA).

From Fig . 14 it may be taken that lipid formulation 01, i. e. the one contai ning 1,2-diphytanoyl- sn-glycere-3-phosphoethanolamine was still effective if the SIRNA content= was 50 nM, whereas lipid formulation 02 containing 1,2-dioleyl—sn-glycero-3-phosphoethanol=amine as helper lipid could gen erate a knockdown of the PTEN omly if the siRNA content was aout 1 pM or more as detected bby a PTEN specific antibody (Santa Cruze, CA). The signal of the unrelated kinase

PRK2 wass used as a loading control and detected by an antibody directed th_ereto.

Example 21: Lipid composition and PEG content

In order to test the impact of PEG on the efficacy on transfection amd delivery of lipid compositicons comprising B-arginyl-2,3-dliaminopropionic acid-N-pall mityl-N-oleyl-amide trihydrochMoride (cationic lipid) as lipid component and 1,2-diphy=tanoyl-sn-glycero-3-

WO- 2005/105152 PCT_/EP2005/004920 phospho ethanolamine (DPhyPE) and 1,2-distearoyl-sn-glycero-3-pho sphoethanolamine- polyethy~lenglycol-2000 (DSPE-PEG2000) the following formulations Weere generated in accordamce with the methods disclosed herein:

GC, - Cs fomulations:

Cationic lipid DPhyPE D&SPE-PEG [mol%] [mol%] [mol%]

H,; — Hs fostmulations:

Cationic lipid DPhyPE DS_PE-PEG [mol%s] [mol%] {mmol %]

I J HJ LA

For any of the aforementioned formulations the Lipid concentration was 1.445 mg/ml, siRNA concentration was 15 uM in 300 mM sucrose. Dilution of the concentrated s—tock-complexes formed yieleded an end concentration of 20, 10, 5 nM siRNA in the cell culture me=dium.

The RNAi molecules contained in said formulations were directed against _PTEN and the sequences ame described in example 22. The lipid formulations were administeread to HeLa cells contained ira a 6 well plate each containing 40,000 cells/well. The cells were analysed for expression of PTEN and thee results depicted in Fig. 15 as Western Blots. p110a e=xpression was used as loading control ancl detected by a monoclonal antmbody specific for p110a_. From any of the Western Blots depicted in Fig. 15 it can be taken that about 1 to 2 mol% of tine helper lipid containing PEG was suitable to provide an efficient knockdown of the PTEN expre=ssion.

It can be concluded that, pr-eferably, the DPhyPE compone=nt is to be replaced by t_he PEGylated helper lipid rather than the cationic lipid component is replaced by the PEGylatec helper lipid.

This can be taken from the above experiment where the EI formulations seem to bes more potent than the C formulations. Thee content of the PEGylated helper lipid is preferably fromm about 0.05 % to 4.9 %, preferably 1 to 3 % and more preferably 2to3 %.

Example 22: In vivo use off an siRNA containing lipid fos rmulation

In order to test the suitability of the siRNA containing lipid formulations according to the present invention, the lipid formulations were used in a mouse model. In contrast to the so-called hydrodynamic pressure injection frequently used to deliver— siRNA to the liver in vivo where a volume corresponding to about 10 % body weight which is. about 2.5 ml of liquid mer mouse is rapidly injected into the tail vein, the present in vivo experi—ments were carried out ssuch that the siRNA containing lipid formmlations were administered systeemically at low volumes= (200 to 300 ul) which were slowly, i. e. over several seconds, injected into the tail vein o f mice thus practising a clinically-relevamt mode of administration. The experimental set up is depicted in

Fig. 16.

Functionally normal rat embryo fibroblasts (RAT2; ATCC :CRL-174) were transfosrmed using oncogenic Ras (Ras''?). The transformed Ras’!2 dependent fibroblasts were swmubsequently injected into mice (6 mice per group; eight-week-old —male Shoe:NMRI-nw/nu1, DIMED,

Germany) which developed a tumor after ten days. At thmis stage, said animals —were either untreated until day 19 after imjection of the transformed fib=roblasts or treatment us ing various formulations was started on day 11. As a further contros] functionally normal mrat embryo fibroblasts were injected into rice which did not develop a tu_mor.

The siRNA molecule which iss referred to herein as T-Ras ceonsisted of a first strand T-Ras 3A having the following sequences: aacguguagaaggcauccu-P in =5’-3’ direction and a second strand

T—Ras 3B having the following sequence: aggaugccuucuacacguu-P ira 5°-3° direction. Please noste that the nucleotides which are printed in bold and which are und=erlined, are 2’-O-methyl nucleotides. At any of the strands, the 3° end starts with a phospha—te depicted by P in the aforementioned sequences.

As a control a PTEN specific siRN_A molecule was designed with a first strand having the fol lowing sequence: 5’ uaaguucuagcuguggngg-P 3° and a second sequence 5’ ccaccacagcuagaacuua-P 3°, whereby the modification pattern is thee same as outlined in corapection with T-Ras 3A and T-Ras 3B, respectively,

The following formulations were admi nistered to the mouse model:

Formulation panel A:

PBS;

T-Ras 3 : 10 mg/kg/atuFect/3.7 mg/kg ; naked T-Ras 3 10 mg/kg ; and

T-Ras 3 5 mg/kg/atuFect 38.5 mg/kg.

Formulation panel B:

PBS; atuFect only 38.5 mg/kg;

PTEN 10 mg/kg/atuFect 38.5 mg/kg; and

T-Ras 3 5 mg/kg/atuFect 38.5 mg/kg. :

Formulation panel C: sucrose (50 mM);

T-Ras 3 3.75 mg/kg/atuFect-PEG 28.9 mg/kg, administered i. v.; anc

T-Ras 3 3.75 mg/kg/atuFect-PEG 28.9 mg/kg, administered i. p. atuFe«ct-PEG as used herein means 50 mol% p-arginyl-2,3-diamino propionic acid-N-palmityl-

N-ole~yl-amide trihydrochloride, 48 mol% 1,2-diphytanoyl-sn-glycero-3-pohosphoethanolamine and 2 mol% 1,2-distearowy}-sn-glycero-3-phosphoethanol_amine-polyethylenglycol ~2000 (DSPE-

PEG2000), in 50 mM sucrose.

The dosage in the animals was 5 mg/kg siRNA and 38.5 rmg/ kg total lipids; the concentration of the components in the injection solution was 0.5 mg/ml siRNA and 3.85 mg/ml total lipids; the molar ratio was: siRNA : 0.5 mg/ml corresponding to 0.04 pmole/m! (molescular weight approximately 12500 Da. The lipid was 3.85 mg/ml ovesrall lipid, whereby the content of the cationic lipid was 1.97 mg/m] (molecular weight 843.6) c=orresponding to 2.3 pmowele/ml cationic lipid. The molar ratio of siFRNA to cationic lipid was 0.0174 to 1.

The results of these expemiments are depicted in Fig. L 7A (formulation panel _A), Fig. 17B (formulation panel B) andl Fig. 17C (formulation panel C) showing the tumor volume as a function of time, i. e. days post cell challenge.

As may be taken from bosth Figs. 17A and 17B the lip=oplexes consisting of T—Ras specific siRNA formulated with a—tuFect show the strongest inkhibition and indicates sspecificity of targeting. It should be note=d that the negative control meolecule PTENV10 does mot show an improved inhibition of tumosr growth when compared to attmFect only (Fig. 17B).

As may be taken from Fig. 17C also atuFect-PEG is highly effective and allows for both i. p. as well as i. v. administration resulting in similar effica cies. In connection the=rewith it is noteworthy that obvious thaat the PEGylated complexes amre functionally active ard it can be assumed that due to PEGsylation such lipid compositio=ns are less toxic than ssimilar lipid compositions which are lack=ing the PEGylated (helper) lipid.

Example 23: Material and mmethods for examples 24 to 2°

Preparation of siRNA-lipopelexes

Cationic liposomes comprising the cationic lipid B-L-argi_nyl-2,3-L-diaminopropio=nic acid-N- palmityl-N-oleyl-amide trihycirochloride, the neutral phospholipid 1,2-diphytanoyl-srm-glycero-3- phosphoethanolamine (Avan=ti Polar Lipids Inc., Alabaster—, AL) and the PEGylate=d lipid N- (Carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl—sn-glycero-3-phosphoeth._anolamine sodium salt (Lipoid GmbH, Ludwigshafen, Germany) in a molar ratio of 50./49/1 were prepared by lipid film re-hydration in 300mM sterile R Nase-free sucrose soluticon to a total lipid concentratiosn of 4.34 mg/ml. Subsequently the mul tilamellar dispersion was further processed by high pressure homogenization (22 cycles at 750 bar and 5 cycles at 1 000 bar) using an

EmulsiFlex C3 device (Avestin, Inc., Ottawa, Canada). The obtained liposo—mal dispersion was mixed with aan equal volume of a 0.5625 mg/ml soRution of siRNA in 300m sucrose, resulting in a calculated charge ratio of nucleic acid backborae phosphates to cationic 1zipid nitrogen atoms of approximately 1 to 4. The size of the lipoplex-dispersion was approxi-mately 120 nm as determined ty Quasi Elastic Light Scattering (NS Submicron Particle Size Analyzer, Beckman

Coulter, Inc. , Miami, FL). For in vitro experiments this dispersion was further diluted to a concentratior of 5-20 nM siRNA in 10% serum containing cell culture medium.

Animal expe-riments

Athymic malee nude mice (Hsd:NMRI-nu/nu, 8 weeks old) were used through-out this study. For tumor theraps, experiments on established tumor xenografts, a total of 5.0 x M0® cells/1 00u! (in the presence eof 50% matrigel for 3Y1-Ras""?) were implanted subcutaneouslwy (s.c.). For tumor therapy exper-iments liposomal siRNA complex solution was administered i.v._ by low pressure, low volume t ail vein injection. Different dosing was achieved by varying irajection schedules (daily vs. bi-~daily) using for a 30g mouse a 200 pl injection volume of a stock solution containing 0.2:8 mg/ml siRNA and 2.17 mg/ml lipid (equivalent to a dose of 1 _88 mg/kg siRNA and 14.5 mg/kg lipid). Tumor volume was determined using a caliper amnd calculated to according the formula volume = (length x width?)/2. All animal experiments in this study were performed according to approved protocols and im compliance with the Suidelines of the

Landesamt fiir- Arbeits-, Gesundheitsschutz und technische Sicherheit Berlira, Germany (No.

G0264/99).

Statistical analysis

Data are expresssed as means + S.E.M. Statistical significance of differences was determined by the Mann-Whitney U test. P values < 0.05 were considered statistically significamnt.

SiRNA-Cy3 up take experiment in cell culture and mice

For uptake stucdies of non-formulated siRNA-Cy3 molecules in cell culture FIeLa cells were incubated with defined amounts of siRNA solution ovemight in serum-free meclium. Uptake of lipoplexed siRMA-Cy3 cells was carried out by tramsfection overnight as me=ntioned below.

”

Treated cells were rinsed with ice ceold PBS and fixed in 4% formmaldehyde/PBS solutiorm for 15 minutes prior to microscopy. To lab el late endosomes and lysosormes, cells were incubate=d with the fluorescent dye LysoTracker~ (Molecular Probes) accomrding to the manufamcturers recommendation and examined bwy confocal miscroscopy aftesr fixation. In vivo d_elivery experiment using fluorescently lazbeled siRNA-Cy3, were carmied out by admini stering formuajated and naked siRNA intrave=nously. Mice were treated with a single 200u] i.v. in-jection at a fi nal dose of 1.88 mg/kg siRNA --Cy3 and 14.5 mg/kg lipid. Milice were sacrificed at cdliefined time-points and fluorescence uptake examined by microscopy on either formalin fixed, p=araffin embedded or OCT mounted frozen tisssue sections.

In vitr-o transfection

Humam HUVEC, Hela, PC-3 cell lignes as well as murine EOMA. and NIH3T3 cell liness were obtained from American Type Cult-we Collection and cultivateed according to the ACC's recomrnendation. Human hepatoma cell line HuH-7 was available at MDC, Berlin. Rat 3Y XX cells expressing oncogenic Ras"? were geraerated by transduction of indumcible Ras¥'? as describesd®®.

Cell Liraes were transfected with siRNJA using the cationic liposommes described above. Briefly, about 1 2 hours after cell seeding diffe=rent amounts of siRNA-lipopMex solution diluted in sserum contain ing medium were added to the mcells to achieve transfection concentrations in a range of 1- 50 nM siRNA. After transfection (48 h) cells were lysed and subjected to immunoblottimng as described”. Following antibodies were used for immunoblotting= Rabbit anti-PTEN (£Ab-2,

Neomarkers), monoclonal p110a/p8=5¥, rabbit anti-PKN3*%, gozt anti-CD31 (Santa Cruz

Biotechnology), rabbit anti-CD34 (Samnta Cruz Biotechnology), rab bit anti-phosphorylated_ Akt (S473) Cell Signaling Technology).

In vivo BrdU assay

To measure cell proliferation in vivo_, mice were treated with BradU (Sigma; 250mg/kgT) by intraperitoneal injection and sacrifice-d two hours later. Formalime fixed paraffin embecided sections of liver or tumor tissue were suzabjected to BrdU staining accoerding to the manufacturers’ protocol (BrdU In situ detection kit, Ph=amingen).

Determimation of microvessel density (MVD)

The num ber of microvessels was determined by counting CD31-/CD34-positive vessels in 3- - 8 randomly selected areas of single turmor sections®. Vessel number as vascular units —was evaluated regardless of shape, branch points and size lumens (referring to “number of vessels”).

Additionally, vasculear density was assessed by determiraation of total length of CID31-/CD34- positive vessel structwures (referring to “sum of vessel lengths”) using the Axiovision 33.0 software (Zeiss). Counting wa.s performed by scanning tumor secti ons at 200x magnification with a Zeiss

Axioplan light microscope.

Histological analysis= and microscopy

After mice were sacrificed, tissues were instantly fixed im. 4.5 % buffered formalin for 16 hours and consequently proccessed for paraffin embedding. 4 ume sections were cut and placeed on glass slides. Tissue section=s were stained with goat polyclonal anti-CD31/PECAM-1 (1:1 000, Santa

Cruz Biotechnology) (alternatively for cryosections rat CD31, 1:100, Pharmingerm) and rat- monoclonal anti-CDZ34 (Cedarlane) to visualize endeothelial cells in paraffin sections.

Immunohistochemistr= and hematoxylin/eosin (H&E) courlterstaining on paraffin tiss_1e sections were performed according to standard protocols. For in vivo uptake studies of flue orescently labeled siRNAs, para ffin sections were directly examin ed by epifluorescence with a Zeiss

Axioplan microscope. Images were recorded and processsed using the Zeiss LSMS imaging software. In depth mic=roscopic analysis of siRNA uptake “was performed with a Zeiss LSM510

Meta confocal microscope. For this, sections were deparaffinized with xylene, rehydrated through graded ethanoel washes, counterstained with Syto=x Green dye (Molecular Pmrobes 100 nM; 10 min), rinsed” and finally mounted in FluorSmve (Calbiochem) for mi croscopy.

Immunofluorescence staining of NIH3T3 cells was performed as described, using following antibodies: the immumnohistochemistry-specific rabbit an ti-phosphorylated-Akt (S473) (Cell

Signaling Technology) and mouse anti-a-tubulin (DM1A, Calbiochem).

Table 1 siRNA sequeneces as used throughout examples 2<¢ to 27 siRNA name sequence 5' tod’

PKN3 s gaagagecuguacugegaga

PKN3 as uccucgeaguacaggeucuc

PTEN s ccsaccacageuagaacuua

PTEN as Lasaguucuageuguggugg

PTEN s (control) CC_accacagcuagaacuua

PTEN as (control) ua_aguucuagcugugouge

PTENs ccaccacageuagaacuua uaaguucuageuguggugg-

PTEN as-Cy3 Cy3

CD31-1s ccaacuucaccauccagaa

CD31-1 as uucuggauggugaaguuge

CD31-2s ggugaunagecccggugpgau

CD31-2 as auccaccggggeuaucace

CD31-6s ccacuucugaacuccaaca

CD31-6 as uguuggaguucagaagugg

CD31-8 s cagaugcucuagaacggaa :

CD31-8 as uuccguucuagapguaucug nucleotides with 2’-O-methyl modifications are under-lined

Example 24: Delivery of naked and formulated siRNAs in vitro and in vivo

In this study, we employed 19-mer siRNA duplexes lacking 3’-overhangs, which are= chemically ~~ stabilized by alternating 2’-O-methy] sugar modificati ons on both strands'®, whereby unmodified nucleotides are faicing modified on the opposite strand. The siRNA molecules actually used are depicted in exampole 23.

In a first step, we analyzed whether these molecules mediate RNAi in cell culture in ®the absence of delivery vehicl es. Immunoblot analysis demonstra-ted that no gene silencing occeurred when naked siRNA was applied at even micromolar concentrations compared to nanomolar concentrations use=d for siRNA-lipoplexes. The results are shown in Fig. 18a.

As may be taken firom Fig. 18 a in more detail, there was a concentration dependent irmhibition of

PKN3 protein expression with lipoplexed siRNAs, but not naked siRNA in Hel _a cells as assesses by immurmoblot. PTEN served as loading control.

We also tested uramodified conventional siRNAs (21-mer, 2 nucleotides 3’-overh=angs)® and several conjugated” molecules including cholesterol-conjugated or peptide-linked siRNAs, but did not detect any target specific reduction of endogenous proteira expression in the absence of delivery vehicles (data not shove).

To analyze whether the lack of gene silencing was the result of an inefficient cellular uptake- due to repulsive effects between thes anionic siRNAs and the negatively charged cell membrane we employed 3° fluorescently (Cy3) labeled siRNAs to study their uprtake by confocal microsceopy.

We, and others have previously’ shown that fluorescence labeling at the 3’end of the antise=nse molecule does not impair RNA silencing activity when transfected with delivery vehicles ®%7.

Surprisingly, we observed a sigriificant uptake of fluorescently labezled siRNAs in the absenc e of transfection reagents when high concentrations of siRNA-Cy3 molecules were applied.

However, the majority of the flimorescence label appeared to end up» in late endosomal/lysoso-mal vesicles as demonstrated by co-localization with the LysoTracker marker suggesting -that unformulated siRNAs remain treapped in the endosomal pathway. In_ contrast, siRNAs transfected as liposomal complexes dissocizated from these vesicles and were released into the cytoplasm.

These results indicate that lipossomal formulation of siRNAs provides at least two benefiacial effects for functional delivery «of siRNAs: an improved cellular uptake and importantly the escape from the endocytotic/enclosomal pathway into the cytoplasm'®, where RNAi-media ted

ImRNA degradation takes place. “The details of Fig. 18b are as fol Jows.

Fig. 18 b shows the intracellulax distribution of naked and formulated siRNAs. Fluorescen_tly

Labeled siRNAs-Cy3 were analyzed by confocal microscopy in Mela cells left and midcile

Panels. Right panels show merged pictures of subcellular distribution after counterstaining with

I_ysoTracker (green; arrows, siRINA-Cy3 localization with respect to the endosomal/lysosonal

Compartment). Upper row, naked siRNA-Cy3; lower row lipoplexed siRNA-Cy3.

T 0 analyze whether the liposormal formulation changes the pharmacological properties of sARNAs in vivo, we injected (low volume and low pressure) a single dose of siRNA-Cxy3 molecules (1.88 mg/kg siRNA) in to the tail vein of mice. Microscopic analysis of several orgamns irxcluding pancreas, lung, kidney. and prostate showed a significaxat increase in Cy3 specific fl vorescence with formulated siRNAs (data not shown). The highest amount of fluorescence was detected in the liver of mice treated with liposomally formulated siRNAs at all analyzed tine points (1h, 4h, 24h post injection, Fig. 18¢). This mresult indicates a better biodistribution of the

SIRNA molecules formulated in lipoplexes when compared to administration of raked siRNAs.

However, the irmproved biodistribution in wholes organs does not necessarrily indicate an intracellular or «ell type specific uptake of the=se molecules, which is a prerequisite for functionality of the delivered siRNAs. A more Cletailed analysis of formulated siRNA-Cy3 uptake in the liver by confocal microscopy revealed that on the cellular level fluorescence staining was preclominantly present in the linings «of the blood vessels and the sinusoids (Fig. 18c, lower panel). A closer inspection of liver vessels revealed that the endothelial layer is clearly labeled bw the fluorescent siRNA-Cy3 in c-ontrast to the PBS control (Wig. 18d, upper row). Inside the endothelial cell, siRNA-Cy3 is exc=lusively present in the cytop_lasm (Fig. 18d, lower panels). The same staining pattern was obse=rved in non-fixed liver cryo sections, which rules out any formmalin fixation artifacts (data not skmown). To test whether fluorescently labeled lipoplexed siRNA also targets the tumor vasculature we treated mice be=aring different experimental tumors with single iv. injections of siRNA-Cy3 lipoplexes . In all three experimental tumor xenografts (two subcutaneoussly, s.c., and one intrahepa tic, i.hep.) we detected significarat fluorescence signals in the tumomr vasculature (Fig. 18e, arrovw). siRNA-Cy3 uptake by the endothelial layer of the tumor vasculature was confirmed by counterstaining with anti-CD34 antibody, an endothelial cell marker (Figs. 18e, lower panels). In addition, uptake of the lipoplex-siRN_A by the endothelium was confirmmed using fluorescently labesled lipids (not shown). Taken togzether, these data demonstrate that «cationic lipid based formulatiz ons of siRNAs improve the kinetic and distribution properties of siR_NAs and allow for a predom-inant uptake of siRNAs into endothelial cells.

The experimental setting for the results shown in Figg. 18c were as follows. Nake=d or Lipolexed siRNA-Cy3 was aciministered by single i.v. injectiora and liver tissue sections of indicated time points were analyzed by epifluorescence microscopy (upper panels). Lower panels, close-up confocal microscopy images of liver sections showing distribution of non-formwulated siRNA-

Cy3 (left picture) compared to lipoplex (right piecture, siRNA-Cy3, red; nuclei, green by counterstaining with Sytox Green). Images were recorded with identical settimngs. Compare staining intensity of liver vessels (arrow) and sinusoids (double arrow).

The details of Fig. 18d are as follows. The endothelizal lining of a liver vessel is Cecorated with fluorescent siRNA~Cy3 (right panel), in contrast to tlhe PBS treated control sectioen (left panel).

Ceonfocal microscopy revealed cytoplasmic delivery of formulated siRNA-Cy3 (red, merged ) in liver endothelial cells (red blood cells, double arrow). No fluomrescence is detectable in the nuacleus (green, arrows).

Tine experimental setting for thie results shown in Fig. 18¢ was as follows. Endothelial cellss of different tumors were targeted with liposomal formulated siRMJAs as indicated by arrows (sL RNA-Cy3, red; nuclei, greem). The upper row shows fluorescent images of sections fr<om subcutaneously grown PC-3 turmor (left panel) and Ras"? transforrrmed 3Y1 rat fibroblast turmaor (nw.iddle panel) or intrahepatically grown HuH-7 tumor (right pamnel). The lower row sho=ws detection of liposome delivered] siRNA-Cy3 in endothelial cells of HuH-7 tumor. The tumor enclothelial cells are shown by H&E staining (left panel) character—ized by their thin cytopla=sm anc the prominent nucleus (axrow). Consecutive sections show corresponding siRNA-CTy3 flueorescence (red, middle panel) and anti-CD34 immunostaining o=f the endothelial cells (right parcel), respectively.

Example 25: Functional delivery of PTEN specific siRNAs to liver and tumor endothelial cells

To demonstrate the ability of siRNA-lipoplexes to silence endogenous gene expression in endothelial cells in vivo, we selected the tumor suppressor PTEN, an antagonist of phossphoinositide 3-kinase (PI 3-kinase), as a target. We intended to monitor functional gerae sileracing of PTEN in a positive read out system by measuring increassed DNA synthesis by BrdlJ inco-rporation in endothelial cell nuclei. Loss of PTEN expressiom is known to chronicall y activate PI 3-kinase signaling, which can be measured by an increas in phosphorylation of th_e dowmnstream kinase Akt'® (Fig. 19a). Chronic activation of PI 3-kinases is also accompanied by am increased rate in DNA synthesis?®.

First_, the RNAI activity of a selected siRNAFTEN (c.f. Example 23), targeting mouse and humar

PTERN mRNA, was verified by lipid-mediated transfection in vitro (Fig. 19a). The identica 1 siRNA sequence carrying 2°-O-methyl modification at every nucleot-ide was used as a negative control (siRNA™"™), since this uniform modification pattern abolishes RNAi activity aompletely'S, PTEN protein knock—down and increased phosphorylation &f Akt was observed by

Emmunoblotting. Immunofluorescemce studies confirmed the enhanced .Akt phosphorylation in the presence of the active siRNA ™ molecule (Fig. 192). This demonstrates the capability of the siRNA"™ molecule to activate PI 3-kinase signaling in cell culture.

To test for PTEN gene silencing in vivo mice (4 per group) were treated ~with either PBS, naked sARNAP™N siRNAP™lipoplex or lipid vehicle on three consecutive days by low pressure, tail vein injection (see Methods). On day four of treatment, BrdU was injected into the mice and two beours later the mice were sacrificed and BrdU incorporation was measured by irmmunohistological staining of liver sections for BrdU positive nuclei. The small size of the erdothelial cells and the difficulties in detecting a reliable signal with phosphorylated Akt and

P"IEN specific antibodies did not allow to detect protein knock-doven in situ. However, consistent with the observed cell specific delivery of fluorescence labeled siRNA to endothelial ce=lls we observed a significant increase in BrdU positive nuclei in the 1i ver endothelium only wikth liposomal siRNAPEN (Fig. 19b). A similar experiment with tumor bearing mice revealed a significant increase as well in the nurmber of BrdU-positive nuclei of the turmor endothelium afier treatment with liposomally formulated active PTEN-siRNA (Fig. 19c)~ The inactive, fully methylated control molecule siRNA“ did not cause an increase in BrdU incorporation relative to the PBS control group. We conclude from these data that stabilized PTEN-specific siFRNAs formulated with cationic lipids are functional in vive to induce gene silencing in encothelial cells after systemic administration,

The details of Fig. 19a are as follows. Transfection of a stabilized PTEN specific siRNA (10

DMD) in vitro reduced PTEN protein level and increased phosphorylation. of the downstream kinaase Akt (P*-Akt) as revealed by immunoblot (right upper panel; PI 3-kirase subunits p110aq,

P85’, unaffected loading control). siRINA*! represents a fully methylated inactive siRNAFTEN mol ecule; ut, untreated cells. Increase of phosphorylated Akt was also visualized by irmnn1unofluorescence staining in NIH3 T3 cells transfected with siRNAPTEN «phophorylated Akt, red; anti-a-tubulin as marker for cell morphology, green).

Fig. 19b depicts representative pictures (upper panels) and corresponding q uantification (lower diag-ram) showing significant differeraces in the number of BrdU positives endothelial nuclei (arrows) in liver samples from anirnals treated with PBS, naked siRINAPTN, lipoplexed

SIRRTAP™N and cationic liposomes, respectively (two pictures shown for each treatment).

Statistical significance: nakesd siRNAF™N vs. siRNA. lipsoplex, *P = 0.0286; liposormes vs.

SIRNA" Jipoplex, *P = 0.40286.

The details of Fig. 19¢ are as follows: Sequence specificity of lipoplexed siRNA'™EN or DNA synthesis was confirmed witt the BrdU assay for the tumor vasculature. Increased BrdU p-ositive nuclei (arrow) were detected in tumor blood vessels (V) from animals treated with siRN_AT™"-

Iipoplex in contrast to siRNA" ipoplex; Tu: tumor tissuee. Quantification of BrdU-peositive —xuclei in endothelial cells wwvas significantly increased: SIRENA®™ [inoplex vs. siRIN.A'TEN- lipoplex *P = 0.032.

MExample 26: In vivo gene silencing of CD31 "Wo demonstrate in vivo siRNAs mediated gene silencing more directly, we focused on targeting a gene selectively expressed ima endothelial cells. We chose platelet-endothelial-cell adhmesion molecule 1 (PECAM-1), also known as CD31, as a suitab Ie target, since its expressi on is restricted to cells of the vascualature system, primarily to endeothelial cells as well as platelets, nonocytes, neutrophils, and se lected T cells? 22,

Sacreening of 2°-O-methyl moclified siRNA molecules (c.f. Exxample 23) in mouse and ht iman derived endothelial cell lines (FIUVEC, EOMA) led to the idenstification of several potent hiaman arad mouse specific CD31-siRRNA molecules (Fig. 20a). The most potent siRNA mole cule,

SIBRNACDE wag liposomallsy formulated as described in example 23 and systemiacally addministered into tumor bearirmg mice for two or for seven damys in a row. Control mice wwere tre=ated with isotonic sucrose sawlution or with lipoplexed siRNAs"™ to test for specificity. After tresatment, mice were sacrificed and gene silencing analyzed in various tissues by real time RT-

PCZR (TaqMan) and immunoblotting.

A mreduction in the CD31 mRN Aw. level in mice treated with SiIRNLAP38_Jinoplex was observe=d in turmnor, liver and lung, but not im spleen tissue samples. The observed reduction in CD31 mR_NA lev-els points to 2a RNAi-mode of action based on mRNA cleavage (Fig. 20b). In additiom, a sigmnificant reduction of CD31 p rotein levels was detected in turmor and liver lysates from mice trezated with siRNA" lipopl exes for two consecutive dayss in contrast to the unchan_ged protein levels observed in the comtrol mice (Fig. 20c, left panel).

Mo test for specificity and equal loading we analyzed in parallel the protein levels of CID34, ayother endothelial cell marker protein, as well as PTEN in these lysates. We have also examined whole cell extracts from spleen and lung, but we did not detect reliable CD31 protein e=xpression by immunoblot analysis im these organs (data not shown_). CD31 protein knock-down was confirmed in an independent experiment on non-tumor bearing mice by seven daily iv. irjections (Fig. 20c, right panel).

Furthermore, the reduction in CD31 expression was also revealed in situ, by measuring dm fferences in the microvessel density (MVD) for the endothelial m=arkers CD31 and CD34 =n a xenograft tumor mouse model. MVD measurement is a surrogate ma-xker for tumor angiogenesis, ard analyzed by immunohistochemicsal staining of blood vessels with CD31 or CD34 spec=ific aratibodies®*?. Formulated CD31 and PTEN specific siRNAs werse administered by tail vein injection with regular volume (200 p11) and regular pressure on two =days in tumor bearing nice (tLamor size 800 mm®). On day three the mice were sacrificed ancl the MVD was compa red be-tween consecutive sections after immunostaining with CD3 1 and CD34 antibod mes, respectively.

Th_e mice treated with the lipoplexed SIRNAP*!® showed a statistic ally significant decrease in the= total amount of CD31 positive vessels as measured by total number of vessels as well as vesssel length (Fig. 20d). Staining with CD34 specific antibodies did not reveal a change in M\~D indicating again specific CD31 silencing. Both control groups, siRNA’ and isotonic sucramse trezated, did not show differences in MVD assessment by either CD31 or CD34 staining. Thnis resmilt along with the molecular data on mRNA and protein knock-d own indicates the specific redmuction in CD31 expression, without 2 decrease in CD34 positive eradothelial cells in respon se to systemic administration of lipoplexed siRNACP'® we conclmuded that in vivo CD! (PESCAM-1) gene silencing can be achieved by administration of cationic lipid formulated siRNAs in the vasculature of tumors and liver.

The details of Fig. 20a are as follows. Fig. 20a shows the identification of potent stabilize=d siRIAs for efficacious CD31 knock-down. HUVEC and murine EOMA cells were transfecte-d withe four different human, mouse specific siRNAs targeting CD31 (CD31-1, -2 ,-6 ,-8) and a control PTEN-siRNA. Specific protein knock-down was assessed by immunoblotting using anti -

CD=1 and anti-PTEN demonstrating highest efficacy of the siRNA®*"- ® molecule.

. oo. . =F £20060 “£7 53

The Fetails of Fig. 20b are as follows. Mice treated on two consecutive days by i.v. injection of lipopBexed siRNA“! showed red-uction of CD31 mRNA levels “in certain tissues as revealed by quasntitative TagMan RT-PCR. The relative amount of CD3L mRNA was normalized to

PTEMN mRNA levels.

The details of Fig. 20c are as followws. CD31 protein knock-down in mice treated systemically with s=iRNACP3 1% Jipoplexes was corafirmed by immunoblot analysis with extracts from liver and tumor- using anti-CD31 antibody a-nd anti-PTEN as well as antl -CD34 (another endothelial marke=r protein) to show equal prote=in loading. Mice were treated by i.v. injection on two (left panel: liver and tumor) or seven consecutive days (right panel: liver». CD31 Protein knock-down was olibserved in the sSiRNA®P!-lipo-plex treated animals in liver ard tumor (see animal 2, left panel) but not in mice treated with isotonic sucrose solution or siRNEAF EN Jipoplex treated mice (see amaimals 1, 3). With a treatment regimen of seven days substanetial CD31 knock-down was observ-ed in animals 5 and 6 in com trast to the control animals 4, 7 and 8 (right panel). The functionality of the siRNAP*".lipoplex used for the in vivo study were verified in parallel in

HUVE-C cells (10 nM siRNA).

The details of Fig. 20d are as follows. In vivo knock-down of CD31 p 1otein was directly assessed by imnunostaining of paraffin tumoxrs sections from corresponding mice treated with isotonic sucroses, siRNA“P*!"%_lipoplex, and siIRNA’ TE. lipoplex. Consecutive= sections were stained with anti-CID31 and anti-CD34 antibodies, respectively, to visualize the twumor vasculature, Reduced staininge intensity for CD31, but not —for CD34, was found in tumor— section from mice treated with si RNAP? "8 lipoplex. MVD quuntification (determined by mumber of vessels, upper diagrarrm, and total lengths of vessel s, lower diagram) of CD31 peositive vessels showing a reduced MVD in the samples from si RNACP? 1 Jipoplex treated mic=e. This difference was not observead by MVD measurement of CID34 positive vessels.

In connestion with the anti-CD31 siRINA molecules disclosed herein it is to be noted that the disclosuzxe of the present application is related to any anti-CD31 siERNA moleclule and more preferab’ly any anti-CD31 siRNA molecule exhibiting the modification pattern shown and describeed herein such as disclosed in conection with the anti CD31-8 siRNA molecule.

WYO 2005/105152 PCT/EP2005/004920

Example 27: Efficacy of s-ystematically administered siBRNAC"! _Jipoplex in tunmor models

In thiss example, we addressed the qeaestion whether formulated siRTNAs against CD31/PECAMN-L-1 exhib t any therapeutic potential on ®umor growth.

CD31 bas been implicated in parti cipating in diverse cellular mechanisms for vessel/plate Tet format=ion and function®#728 but its potential contribution to neo-vascularization during tum-or growtln has not been addressed so fax. The siRNA molecules chosem. for the therapeutic approach comprised the specific siRNAP*!® and siRNAFTEN a5 a control moRecule. The siRNA“P*' 8. ard siRNA_F®N lipoplexes for the in wivo efficacy studies were tested in a dose dependent transfeaction experiment in HUVEC prior to the in vivo experiment. Representative immunoblo-ts demonstrating the functionality and peotency of these siRNA-lipoplexxes are shown in Fig. 21a.

Knock—down of CD31 protein was achieved with SIRNAS! in fie Tow sub-nanomolar rang=e with these formulations. Specificity of the siRNA? med iated gene silencing waxs demonsstrated by probing for PTEN, phosphorylated Akt and CD3<1. Unlike transfections withh siRNA" ™, the phosphorylation statis of Akt was not affected in HRUVEC cells by reduction imn

CD31. €D34 protein level was not changed with both lipoplexes when compared to untreatec] controls . The potential therapeutic effesct of the systemically administered CD31-siRNA-lipoplex< was investigated in mice bearing two clifferent types of s.c. tumor xen _ografts.

First, we established a regimen whicka allowed for repeated systemi_c treatment using different lipoplex daily doses. Different total doses were achieved by administration of daily or bi-daily=~ tail vein injections of 200ul lipoplex solution (single dose 1.88mg/Ecg/d siRNA; 14.5 mg/kg/d lipid). W/e did not observe severe toxic effects on the animal health status as assessed by monitorimng changes in body weight as zn overall marker of general health (Fig. 21b).

Subseque=ntly, we analyzed the two dosing regimens representing edther daily or bi-daily i.v. treatments in an efficacy study of sIRNAP*! finoplex on tumor growth inhibition. Both treatment: regimens resulted in a clear inhibitory effect on tumor growvth of an established, fast growing 3Y1-Ras""® sc. xenograft with lipoplexed siRNACD?S (Fi_g. 21c). Notably, for this particular- tumor xenograft the bi-daily regimen improved the inhibitory effect on tumor growth.

This inhibition was statistically significant when compared to the siRNA ™N_lipoplex ams well as the sucrose treated control groups (Fig. 21c).

In an additional experiment, systemic treatment of a slowver growing s.c. PC-3 tumor »xenograft with liposomal formulated siRNACP3!-8 similarly caused a significant delay in tumor growth in contrast to the siRNA"™ control (Fig. 21d). Taken together, the in vivo xenograft experiments clearly demonstrate that growth of tumor cells in nude mice can be suppressed by systemic administration. of liposomal formulated CD31-siRNAs. These data also imply tha t CD31 (PECAM-1), a non-classical drug target, appears to be a suitable target for RNAi bassed anti- angiogenic therapeutic intervention.

The details of Fig. 21a are as follows. Quality control and efficacy testing of lipoplexed_ siRNA used for systemic tumor treatment in HUVEC, Immunoblotting using anti-CD31 antibody revealed a comvcentration dependent knock-down of CD32 in the case of siRNA? 8 but not with control siRNA". Reduction of CD31 had no effect on PI 3-kinase signaling as resvealed by monitoring _Akt phosphorylation status (P*-Akt), in contrast to the siRNA" control _ CD34 protein level was not affected.

The details of Fig. 21b are as follows. The influence of two different siRNA-lipoplex doses on body weight was monitored. Different SIRNA". Jipop lex doses (squares: daily in—jection resulting in 1.88 mg/kg/d siRNA and 14.5 mg/kg/d lipid; di amonds: bi-daily injection (8h apart), 3.75mg/kg/d siRNA and 28.9 mg/kg/d) were administered for seven consecutive days, and changes in body weight were measured and plotted as mean value (n = 7 mice). For compzaarison, body weights (rmean + S.E.M.) of animals treated with isotonic sucrose solution (circle=s) are shown.

The details of Figs. 21c and 21d are as follows. Inhibition of tumor growth by CD31-silRNA- lipoplex treatmemt. Two different tumor xenografts (c: 3YX1-Ras¥'?, d: PC-3) were estabHished s.C. in nude mice (c: left diagram: n = 8 mice per group, right, n = 7 mice per group; d: n= § per group). Mice bearing tumors were treated with siRNA CD?! ~P.lipoplex (diamonds), siRNA TEN.

Iipoplexes (triangles) or isotonic sucrose (solid spheres). Different treatment regimens were applied as indicated; single arrow, daily; double arrows, bi-daily. (c) Growth of established 3Y1-

Ras" tumors was significantly inhibited by siRNA“™'lipoplex when compare d to siRNA" _lipopLexes by applying the bi-daily dosing regimen (right diagram). (d) Growth of established PC-3 xemografts was significantly inhibited with siRNA “®*"®_[ipopolex in comparison to siRNA"™_Lipop-lex treated administered as jmdicated (1.88mg/kg/d siRMNA; 14.5 mg/kg/d lipid; arrow). Data mrepresent the means + S.E.M. 5 significance: *P < 0.05 ac=cording to Mann-

Whitney.

The following references were are inherent to examples 23 to 28 and are incorporated herein in their entirety by refexrence: 6. Elbashir, S.MIL. et al. Duplexes of 21-nucleot-ide RNAs mediate RNA inte=rference in cultured mamr malian cells. Nature 411, 494- 8 (2001). 16. Czauderna, F. et al. Structural variations anc. stabilising modifications of synthetic siRNAs in maemmalian cells. Nucleic Acids Res 31, 2705-16 (2003). 17. Chiu, Y.L. & IRana, T.M. RNAi in human cells: basic structural and func=tional features of small interferimng RNA. Mol Cell 10, 549-61 «2002). 18. Zelphati, O. & Szoka, F.C., Jr. Mechanism of oligonucleotide release frorn cationic liposomes. Proc Natl Acad Sci US 4 93, 114-93-8 (1996). 19. Stambolic, V. «et al. Negative regulation of PEXB/Akt-dependent cell survi—val by the tumor suppressor PTIEN. Cell 95, 29-39 (1998). 20. Klippel, A. et al. Activation of phosphatidylirositol 3-kinase is sufficient _for cell cycle entry and prommotes cellular changes characteristic of oncogenic transform _ation. Mol Cell

Biol 18, 5699-711 (1998). 21. Watt, S.M., Gs<chmeissner, S.E. & Bates, P.A _. PECAM-1: its expression a=nd function as a cell adhesion molecule on hemopoietic and «endothelial cells. Zewk Lymp~homa 17, 229- 44 (1995). 22, Newman, P.J. est al. PECAM-1 (CD31) clonin_g and relation to adhesion meolecules of the immunoglobulim gene superfamily. Science 2487, 1219-22 (1990). 23. Ilan, N. & Madwi, J.A. PECAM-1: old friend, mnew partners. Cpr Opin Cel”l Biol 15, 515- 24 (2003). 24. Fox, S.B. & Hamris, AL. Histological quantita-tion of tumour angiogenesis. Apmis 112, 413-30 (2004).

25. Uzzan, B,, Nicolas, P ., Cucherat, M. & Perret, G.Y. Micreovessel density as a prognostic factor in women with breast cancer: a systematic review of the literature and meta- analysis. Cancer Res «64, 2941-55 (2004). 26. Weidner, N., Semple, J.P., Welch, WR. & Folkman, J. Twumor angiogenesis and metastasis-—-correlatiom in invasive breast carcinoma. N EraglJ Med 324, 1-8 (1991). 27. Ian, N,, Mahooti, S. &: Madri, J.A. Distinct signal transdiaction pathways are utilized during the tube formation and survival phases of in vitro amugiogenesis. J Cell Sci 11 ( Pt 24), 3621-31 (1998). 28. Solowiej, A., Biswas, P., Graesser, D. & Madri, J.A. Lack™ of platelet endothelial cell” adhesion molecule-1 attenuates foreign body inflammatiomn because of decreased angiogenesis. 4m J Pathol 162, 953-62 (2003). 38. Leenders, F. et al. PKI™N3 is required for malignant prostate cell growth downstream of activated PI 3-kinase. £Embo J 23, 3303-13 (2004). 39. Klippel, A., Escobedo, J.A., Hirano, M. & Williams, L.T. “The interaction of small domains between the subunits of phosphatidylinositol 3-ki-nase determines enzyme activity. Mol Cell Biol 14, 2675-85 (1994). =40. Santel, A. & Fuller, M_T. Control of mitochondrial morphology by a human mitofusimn. J

Cell Sci 114, 867-74 (2001).

Whe features of the present FAnvention disclosed in the specifiacation, the claims and/or the

Glrawings may both separately, and in any combination thereof be material for realizing the imnvention in various forms thereof.

Claims (61)

720067 08457 Claims

1. A compound according to formuala (J), O R1 hs R2 NH,+ oO wherein R; and R; are each and independently selected from tlhe group comprising alkxyl; n is any integer between 1 and 4; R; is an acyl selected from the group comprising lysyl, ormithyl, 2,4-diaminobutywryl, histidyl and an acyl moiety according to formula (II), i H AE NH, m hid M NHL + NH, + v @m wherein m is any integer from 1 to 3 and Y" is a pharmaceutically acceptab le anion.

2. . The compound according to claim 1, wherein R; and R; are each and independen®ly selected from the group comprising lauryl, myristyl, palmityl and oley™1.

3. The compound according to any of claims 1 and 2, where in R; is lauryl and Ra is my~zistyl; or

NO 2005/105152 PCT/EP2005/004920 —R| is palmityl and R; is oleyl.

4. “The compound according to any of claims 1 to 3, wherein m is HM or 2.

5. "The compound according to amy of claims 1 to 4, wherein th-e compound is a cationic lipid, preferably in association with an anion Y". :

6. “The compound according to any of claims 1 to 5, wherein Y" i s selected from the group comprissing halogenids, acetate and trifduoroacetate.

7. "The compound according to any of claims 1 to 6, wherein the caompound is selected from the group comprising - B-arginyl-2,3-diamino propionic acid-N-palmityl-N-oley=-amide trihydrochloride IES NS SN HOT NA+ pn HEN CI Cr NH, + Ci - B-arginyl-2,3-diamino propionic acid-N-lauryl-N-myristy~l-amide trihydrochloride Fh Vd a ol GP Pn dh + + H,C Ct cr NH, + Cr and - e-arginyl-lysine-N-lauryl -N-myristyl-amide trihydrochlor-ide

Cr + H, RR Lo SPUN NEL+ lad 2 OPN NES+ EN NH “ HC or 0

8. A composition comprising as a lipid component a_ compound according to ary of claims 1 to 7, and a carrier.

9. The composition according to claim 7, whereir the composition comprises a further constituent.

10. A pharmaceutical composition comprising a compwound according to any of claims 1 to 7 and a pharmaceutically active compound and preferably a pharmaceutically acceptabmle carrier.

11. The composition according to any of claims 8 to 1 0, wherein the pharmaceut=ically active compound and/or the further constituent is selected rom the group comprisitag peptides, proteins, oligonucleotides, polynucleotides and nucleic aci ds.

12. The composition according to claim 11, wherein thhe protein is an antibody, poreferably a monoclonal antibody.

13. The composition according to claim 11, wherein the nucleic acid is selecte=d from the group comprising DNA, RNA, PNA and LNA.

14. The composition according to any of claims 11 or 13, wherein the nucleic acid is a functional nucleic acid, whereby preferably the functional mucleic acid is selected fron the group comprising RNAI, siRNA, siNA, antisense nucleic acid, ribsozymes, aptamers and spie-gelmers.

15. The composition according to any of claims 8 to 14, further comprising at= least one helper lipid component, whereby preferably the helper ligpid component is selected from the group comprising phospholipids and steroids.

16. The composition according to claim 15, wherein the helper lipid component is selected frorm the group comprising 1,2-dipkytanoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleyl- sn-glycero-3-phosphoethanolamine.

17. The composition according ®o any of claims 15 to 16, wherein the content of the helper lipic component is from about 20 rmol % to about 80 mol % of the overall lipid content of the commposition.

18. The composition according to claim 17, wherein the commtent of the helper lipid component is from about 35 mol % to about 65 mol %.

19. The composition according to any of claims 16 to 18, wherein the lipid is B-arginyl-2,3- diammino propionic acid-N-palmityl-IN-oleyl-amide trihydrochloride, ard the helper lipid is 1,2- dipb-ytanoyl-sn-glycero-3-phosphoetiaanolamine.

20. The composition according to claim 19, wherein the lipid is 50 rmol% and the helper lipid is 50 mol% of the overall Lipid contemmt of the composition. ‘

21. The composition according to any of claims 8 to 20, wherein the composition contains at least ®wo helper lipids.

22, The composition according toe claim 21, wherein at least one Thelper lipid comprises a moiety which is selected from the group comprising a PEG moiety, a HEG moiety, a polyhzydroxyethyl starch (polyHES) moiety and a polypropylene moiety, whereby such moiety preferably provides a molecule weight= from about 500 to 10000 Da, moxe preferably from about 2000 0 5000 Da.

23. The composition according to claims 21 or 22, wherein the helper lipid comprising the PEG moiety is selected from the group comprising 1,2- distearoyl-sn-glycero-3- phosplaoethanolamine, 1,2-dialkyl-sn-gzlycero-3-phosphoethanolamine; arad Ceramide-PEG

24, The composition according to claim 23, wherein the PEG moiety has a molecular weight from a bout 500 Da to 10000 Da, preferably from about 2,000 to 5,000 Da, more preferably a molecumlar weight of 2,000 Da.

25. The composition according to claim 24, wherein thme composition comprises as the lipid component -arginyl-2,3-diamino propionic acid-N-palmit—yl-N-oleyl-amide trihydrochl<cride, as a first helper lipid 1,2-diphytanoyl-sn-glycero-3-phosphoesthanolamine and as a secon_d helper lipid 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-PEG 2000.

26. The composition according to claim 25, wherein the content of the second helpex lipid is from about 0,05mo01% to 4,9 mol%, preferably about 1 to 3 mol%.

27. The composition according to claim 26, wherein the content of the lipid is from 4-5 mol% to 50 mol%, the content of the first helper lipid is from 45 to 50 mol% and, under the proviso that there is a PEGylated second helper lipid, the content of the second helper lipid is fromm about 0,1 mol% to about 5 mol %, preferably from about 1 to 4 meol% and more preferably about 2 % , whereby the sum of the content of the lipid, of the lipid, of the first helper lipid and of the= second helper lipid is 100 mol% and whereby the sum of the first he=lper lipid and the second helper lipid is 50 mol%.

28. The composition according to any of claims 21 to 27 containing: a) 50 mol% of P-arginyl-2,3-diamino propicenic acid-N-palmityl-N-oleyB-amide trihydrochloricie, 48 mol% of 1,2-diphytanoyl-sn-glycero-3-pho=sphoethanolamine; and 2 mol% 1,2-di stearoyl-sn-glycero-3-phosphoet-hanolamine-PEG2000. ~or b) 50 mol% of B-L-arginyl-2,3-L-diamino propionic acid-N-palmityl-N-oleyl—amide trihydrocloride, 49 mol% 1,2-diphytanoyl-sn-glycero-3-phosph_octhanolamine; and

We 2005/105152 PCT/EP>2005/004920 1 mol% N(Carbonyl-metho xypolyethylenglycol-2000)-1,2-distearoyl-sn-glycero- 3-phosphoethanolamine, preferably the sodium salt thereof.

29. "Ahe composition according to any o f claim 8 to 28, wherein the functional nucleic acid is a double-stranded ribonucleic acid, wherein the composition further comprises a nucleic acid, preferabely a functional nucleic acid which is more preferably a double-stranded ri bonucleic acid and mosst preferably a nucleic acid selected from the group comprising RNAi, siRNA, siNA, antisenses nucleic acid and ribozyme, whereby preferably the molar ration of RNJAi to cationic lipid is —from about 0 to 0.075, preferably from about 0.02 to 0.05 and even m ore preferably

0.037.

30. The composition according to any of claims 8 to 29, wherein the compound and/or the helper lipid component is present as a dispersion in an aqueous medium.

31. Tihe composition according to any of claims 8 to 29, wherein the compound and/or the helper lipid component is present as a solutdon in a water miscible solvent, whereby preferably the solvemnt is selected from the group comprising ethanol and tert.-butanol.

32. Tine composition according to any of” claims 8 to 31, wherein the functional nucleic acid is a double-stranded ribonucleic acid, preferably a nucleic acid selected from the group comprisirmg RNAI, siRNA, siNA, antisense mucleic acid and ribozyme, and whereby preferably the molar ratio of RNAI to cationic lipid is firom about 0 to 0.075, preferably from about 0.02 to

0.05 and even more preferably 0.037

33. Th_e composition according to any of claims 8 to 32, preferably 29 to 32, wherein the composition contains a nucleic acid, whereby the charge ratio of nucleic acid backbone phosphatezs to cationic lipid nitrogen atoms is about from 1: 1,5 — 7, preferably 1: 4.

34. Thee composition according to any of claims 8 to 33, preferably 29 to 33, wherein the size of the particles in the dispersion is about 120 rim.

35. Thes composition according to any of claims 8 to 34, preferably 29 to 34, wherein the dispersion is a stock dispersion containing about 1 to 100 HM siRNA, whereby preferably the stock dispersion is diluted in vivo or in vitro by 1: 100 to 1:10000, more preferably 1 = 1000.

36. Use of a compound according to any of claims 1 to 7 or a composition according to Zany of claims 8 to 35, for the mammufacture of a medicament, preferably for the treatment of canecer and/-or cardiovascular related diseases.

37. Use according to claizm 36, wherein the medicament is =for the treatment of cancer, whemreby preferably the cancer is selected from the group comprisirmg solid and non-solid tumeors and whereby more preferably the solid tumor is selected from the group comprising pancreatic canc=er, breast cancer, prostate «cancer, lung cancer, colon cancer and. hepatocellular carcinoma.

38. Use according to claim 35 or 36, wherein the cancer involvess a process selected from athe group comprising angiogenesis and neoangiogenesis.

39. Use according to claim 36, wherein the medicament is for a dministering the nucleic aecid toa cell selected from the group comprising endothelial cells, epithelial cells and tumor cells, preferably the cell is an endoth elial cell.

40. Use according to claim 39, wherein the endothelial cells are endothelial cells of vasculature.

41. Use according to claim 40, wherein the vasculature is vasculature arising from neoa-mgiogenesis, preferably tuxmor associated neoangiogenesis.

42. Use according to claims 40, wherein the vasculature is sclected from the gro-up comprising liver vasculature, hneart vasculature, kidney vasculature, pancreactic vasculature a nd lung vasculature.

43. Use according to any of claims 36 to 42, wherein the mmedicament is for systemic administration.

44. Use according to any” of claim 36 to 42, wherein thes medicament is for local administration.

45. Use according to any of claims 36 to 44, wherein the medicament is a diagnostic agent.

46. Use o-fa compound according to any o»f claims 1 to 7 or a composition according to any of claims 8 tc 35, as a transferring agent.

47. Use according to claim 46, wherein the transferring agent transfers a pharmaceutically active compo-nent and/or a further constituent into a cell, preferably a mammalian cell and more preferably a bmuman cell.

48. Use according to claim 47, whereby th e cell is an endothelial cell, preferably a vascular associated endothelial cell.

49. A me=thod for transferring a pharm aceutically active compound and/or a further constituent irato a cell or across a membrame, preferably a cell membrane, comprising the following steps: : - providing the cell or the membrane; - providing a compound accordings to any of claims 1 to 7; - providing the pharmaceutically active compound and/or the further constituent; and - contacting the cell or the membrane with the pharmaceutically actisve compound and/or the further constituent, arad the compound according to any of claims 1 to

7. :

50. A metthod for transferring a pharmaceutically active compound and/or a further constituent insto a cell or across a membrarme, preferably a cell membrane, providing the following stepss: - _providing the cell or the membrane; - —providing a composition accordirag to any of claims 8 to 35; and

- contacting the cell or the membrane with the compositiora according to any of claims 8 to 35.

51. The method according to claims 49 and 50, wherein the ph=amaceutically active compound comprising as further step: - detecting the pharmaceutically active compound and/or the further constituent in the cell and/or beyond the mermbrane,

52. A me=thod for the synthesis of N-palm ityl-oleylamine comprising thes following steps: - providing oleic acid; - providing palmitylamine; - reacting the oleic acid and thme palmitylamine to form N-poalmityl-oleoylamide; and - reducing the N-palmityl-oleoyl amide to N-palmityl-oleylamire, whereby the eleic acid is at least 90 %, more preferably 95 % and most preferably 99 % pure, whereby the gpercentage is the molar ratio of oleic acid and any fatty acid different from oleic acid.

53. The nmethod according to claim 52, ~wherein the oleic acid and thme palmitylamine are reacted at roomn temperature.

54. The mmethod according to claim 52 or 53, wherein the oleic acid is subject to a pre- treatment prior to reacting it with the palmnitylamine, whereby the pre-—treatment comprises reacting the oleic acid with ethylchloroformaate, preferably in anhydrous dichloromethane or anhydrous tetr—ahydrofuran.

55. The mesthod according to any of claim s 52 to 54, wherein the reactioen is performed at 0° C, preferably v.ander inert gas.

56. The method ac=cording to claims 54 and 55, whereby the reaction is further resacted with an acid scavenger, wh ereby the acid scavenger is prefacrably selected from the group «comprising triethylamine, diisopraspylethylamine and pyridine.

57. The method acecording to any of claims 52 to 5¢5, whereby the molar ratio of chaloroformic acid ethyl ester, oleic amcid, triethylamine and palmityla_ nine is about 1-1.05: 1:1 : 1-33 : 1-1.10.

58. The method according to any of claims 52 to 57, wherein the reduction of the N-palmityl- oleoylamide to N-palrmity-oleylamine is performed usimng LiAIH,.

59. The method acecording to any of claims 52 to 57, wherein upon reacting the oleic acid with the palmitylaminee, the reaction is washed, precipitated and the precipitate thus obtained optionally re-crystallisesd.

60. Use of a compomund according to any of claims 1 to 7 or a composition accord ing to any of claims 8 to 35 for sy_stemic administration, preferabl=y systemic administration to a vertebrate.

61. Use according: to claim 60, whereby the vertebrate is a mammal, more preferably a mammal selected from the group comprising mouse, ra®t, guinea pig, cat, dog, monkey snd man.