WO2008043366A2

WO2008043366A2 - Three-domain compounds for transmembrane delivery

Info

Publication number: WO2008043366A2
Application number: PCT/DK2007/050149
Authority: WO
Inventors: Uffe Koppelhus; Peter Eigil Nielsen
Original assignee: Københavns Universitet
Current assignee: Københavns Universitet
Priority date: 2006-10-13
Filing date: 2007-10-15
Publication date: 2008-04-17
Anticipated expiration: 2009-04-13
Also published as: WO2008043366A3

Abstract

The present invention is based on the finding that the transmembrane delivery of a biological agent coupled to a cationic domain can be further improved by coupling to a lipophilic domain, such as a fatty acid. Thus, one aspect of the present invention is three-domain compounds with improved transmembrane delivery. Other aspects are use of the three-domain compounds for therapy, pharmaceutical compositions comprising the three-domain compound, as well as methods for improving transmembrane delivery.

Description

Three-domain compounds for transmembrane delivery

Background of the invention

If a molecule (e.g. a therapeutic agent) is to reach an intracellular target it is required to travel through an aqueous environment and to transgress the non- polar lipid bi-layer of the cell membrane, respectively. Only a small subset of molecules possesses a solubility profile which accommodates both these requirements. Consequently, the majority of compounds (those which are too polar to passively diffuse through the cells non-polar lipid membrane and those too non-polar to be easily formulated and distributed in the aqueous milieu) have low bioavailability and thus low therapeutic value per se. Examples include most small, organic molecules and biopolymers such as proteins, peptides, DNA oligomers, DNA analogue oligomers, siRNAs and plasmids. To overcome the restrictions posed by low bioavailability, a lot of resources have been invested in the search for vehicles with a general capacity for transporting "macromolecules" to the interior of cells. However, cellular and systemic delivery of larger (mw > 1000) molecules and drugs is still a challenge in medicine despite much effort. These efforts have among other candidates resulted in the identification of a class of peptides, called cell-penetrating peptides (CPP), with a capacity to cross biological membranes. The class of CPP includes drosophila transcription factor derived pAnt (penetratin) herpes simplex virus type-1 transcription factor V22, Tat peptide, Tatp from HIV-I transactivator, Tat, polyarginine and the galanin/mastoparan chimera, Transportan. The cell penetrating (cationic) peptides were long considered a possible solution to this problem of delivery of macromolecules, but recent insight has clearly shown these not to be optimal. In particular it has been found that their main route of entry into cells is through an endosomal pathway, which does not deliver the drug molecule to the appropriate cellular compartment, the cytoplasm and/or the nucleus, and therefore significantly reduces efficacy. Much controversy also exists concerning the relative potency of these peptides and their mode of action. The mechanism by which they act is therefore considered to be unknown. Furthermore, most - if not all - cationic peptides suffer from side effects relating to cytotoxicity necessitating a tight rope balance between delivery and toxicity. Also, the most effective peptides are typically larger than the drug cargo and/or have to be conjugated to the drug through a cleavable linker (e.g., a disulfide bridge) thereby greatly complicating the synthesis of the drug conjugates. Finally, no improvements of the activity have been introduced to any of these peptides as a result of theoretical considerations based on a hypothesis. Therefore, effective, easily accessible drug carriers are still needed.

Summary of the invention

It is well known in the art that transmembrane delivery of biological agents can be improved by coupling to a cationic domain, such as a cell penetrating peptide. The present invention is based on the finding that the transmembrane delivery of a biological agent coupled to a cationic domain can be further improved by coupling to a lipophilic domain, such as a fatty acid. Thus, one aspect of the present invention is three-domain compounds with improved transmembrane delivery. Other aspects are use of the three-domain compounds for therapy, pharmaceutical compositions comprising the three-domain compound, as well as methods for improving transmembrane delivery.

Detailed description of the invention

A first aspect of the present invention is a compound comprising :

a. a modulator domain capable of modulating the activity of a cellular component b. a cationic domain and c. a lipophilic domain

-wherein the cationic domain increases the transmembrane delivery of the modulator domain and,

-wherein the lipophilic domain increases the transmembrane delivery of the modulator domain coupled the cationic domain.

As used in the present context, a domain refers to a particular part of the compound that is structurally and/or functionally distinct from the other parts of the compound. Thus, the term domain as used herein is not limited to a protein domain, although the domains of the compound may be proteins or peptides. The above described compound is also herein referred to as a three-domain compound.

A modulator domain refers to a domain that is capable of modulating the activity of a cellular component or in other words, a biologically active agent that act inside a cell. Thus, the modulator domain may e.g. be an antisense molecule capable of modulating the activity of a cellular nucleic acid, as will be further outlined below, along with other embodiments.

A cationic domain refers to a domain that has a net positive charge.

A lipophilic domain refers to a domain that has a poor solubility in water.

Lipophilicity

In a preferred embodiment, the lipophilic domain is characteristic in that it is more lipophilic than 1-hexanol.

In another preferred embodiment, the lipophilic domain is characteristic in that it is more lipophilic than hexanoic acid.

When referring to the lipophilicity of the lipophilic domain, what is meant is the lipophilicity of the lipophilic domain separated from the three-domain compound, e.g. a free fatty acid if the lipophilic domain is a fatty acid that has been conjugated to a lysine.

The lipophilicity of a compound may be expressed in terms of the octanol-water distribution coefficient of the compound, log(D) = log(concentration in octanol/concentration in water). Note, that ionized as well as non-ionized (neutral) species are used for calculation of the distribution coefficient, i.e. log(D) is dependent on pH.

In a preferred embodiment, the lipophilic domain has a log(D) value at pH 7 that is the same or is larger than the log(D) value of hexanoic acid. In another embodiment, the log(D) value at pH 7 is larger than the log(D) value selected from the group consisting of: the log(D) value of pentanoic acid, the log(D) value of heptanoic acid, the log(D) value of octanoic acid, the log(D) value of nonanoic acid and the log(D) value of decanoic acid.

The lipophilicity of a compound may also be expressed in terms of the octanol- water partition coefficient of the compound, log(P) = log(concentration in octanol/concentration in water). Note, that only the neutral species are used for calculation of the partition coefficient, i.e. log(P) is independent of pH.

In a preferred embodiment, the lipophilic domain has a log(P) value that is that same or is larger than the log(P) value of 1-hexanol.

In another embodiment, the log(P) value is larger than the log(D) value selected from the group consisting of: the log(P) value of 1-pentanol, the log(P) value of 1- hexanol, the log(P) value of 1-octanol, the log(P) value of 1-nonanol and the log(P) value of 1-decanol.

In another embodiment, the lipophilic domain (when part of the three-domain compound) decreases the solubility of the three-domain compound in water, as compared to the same compound without the lipophilic domain.

Thus, in a preferred embodiment, the lipophilic domain is characteristic in that it decreases the solubility of the three-domain compound in water, as compared to the same compound without the lipophilic domain by a factor selected from the group consisting of: at least a 2-fold decrease, at least a 5-fold decrease, at least a 10 fold decrease, at least a 20 fold decrease, at least a 50 fold decrease, at least a 100 fold decrease, at least a 500 fold decrease, at least a 1.000 fold decrease, at least a 10.000 fold decrease, at least a 50.000 fold decrease, at least a 100.000 fold decrease, at least a 500.000 fold decrease and at least a 1.000.000 decrease.

Further, the lipophilic domain of the present invention increase the transmembrane delivery of the modulator domain coupled to the cationic domain. Whether a lipophilic domain fulfils this criteria can be determined e.g. using the methods outlined in the examples section.

Transmembrane delivery in the present context refers to transport of a compound from one side of a biological membrane to the other side of the membrane. A very preferred embodiment of transmembrane delivery is transport across the cell membrane of a cell, such that after transport, the compound resides inside the cell. Note that no reference is made to the mechanism of transport. Thus, the compound may still reside within a lysosome after having crossed the outer membrane, i.e. it crosses the cell membrane by way of endocytosis. The compound may also cross the membrane by active transport or diffusion through the membrane. However, the present invention does not relate to the mechanism, but only to the end result, namely improved transmembrane delivery as compared the same compound without the lipophilic domain.

In a preferred embodiment, the modulator domain, the cationic domain and the lipophilic domain are three distinct domains. That is to say that one domain is not functioning both as e.g. modulator and lipophilic domain. Furthermore, when referring to three distinct domains, it is important to note that these are preferably covalently coupled. I.e. a modulator domain coupled to a cationic domain formulated in a lipid formulation is not encompassed by the claims.

As outlined below, in one embodiment, the modulator domain need not necessarily be covalently coupled the cationic domain and the lipophilic domain. The modulator domain may be bound by a modulator binding domain which in turn is covalently coupled to the cationic domain and the lipophilic domain.

Further in a preferred embodiment, the three-domains together make up more than 80% of the total molecular weight of the compound. I.e. linkers and other moieties make up less than 20% of the total molecular weight of the compound.

The modulator domain is preferably selected from the group consisting of polypeptides, nucleic acids and derivatives and mimics thereof. The meaning of the words "derivatives" and "mimics" will be further detailed with below with regards to nucleic acids and polypeptides.

The modulator domain - nucleic acids Thus, in a preferred embodiment, the modulator domain is a nucleic acid selected from the group consisting of: an oligonucleotide, a double stranded DNA, an antisense molecule, a siRNA, a miRNA, a ribozyme, a triplex forming oligonucleotide, an aptamer or a plasmid. The modulator domain may also be a minor groove binding polyamide.

As is well known to the skilled man, such nucleic acids can modulate the activity of a cellular target. For some of these nucleic acids, they may be delivered with a complementary counterpart that is not covalently coupled to the compound. E.g. when delivering a siRNA, the siRNA will typically be a double stranded RNA with strands of 18-22 nt. In this case, it may be the passenger strand which is coupled covalently to the compound, and the antisense strand is then hybridised to the passenger. Such compounds are also encompassed by the scope of the invention, as the siRNA is viewed as a complex with two strands of which one is covalently coupled to the compound. The same applies to other double stranded nucleic acids.

Thus, in a preferred embodiment, the modulator domain is base paired to a nucleic acid (serving as a modulator binding domain), which in turn is covalently coupled to the lipohilic domain and the cationic domain. Specific examples of this embodiment are e.g. a double stranded siRNA with an overhang, said overhang being base paired to a nucleic acid (modulator binding domain), that is covalently coupled to the lipophilic domain and the cationic domain. Also a microRNA, aptamer etc. may be base paired to a nucleic acid that is covalently coupled to the lipophilic domain and the cationic domain. Preferably, the region of base pairing does not interfere with the activity of the modulator domain (microRNA, aptamer, siRNA etc. ). In a preferred embodiment, base pairing between the modulator domain and the modulator binding domain is reversible and preferably of less than 15 base pairs, such as less than 12 or less than 10 base pairs. The same arguments apply to delivery of other double stranded nucleic acids, e.g. plasmids where only one strand is covalently coupled to the complex.

Preferred embodiments of the nucleic acid and its analogues and mimics are DNA, RNA, LNA, PNA, morpholino or any combinations thereof. Further nucleotide analogues/mimics capable of sequence specific base pairing may also be comprised within the nucleic acid. In other embodiments, also nucleotide analogues/mimic not capable of sequence specific base pairing is included. One such example is the so-called universal bases that fit into a Watson-crick helix adjacent to any of the natural bases.

A particular preferred nucleic acid mimic is PNA (peptide nucleic acid).

Modulator domains embodied as nucleic acids are preferably between 6 and 40 monomers. I.e. if the nucleic acid is DNA, it is preferably between 6 and 40 nucleotides long. In other preferred embodiments, the nucleic acid has a length counted in monomer numbers selected from the group consisting of: 6-10, 10-14, 14-18, 18-22, 22-26, 26-30, 30-34, 34-38, 38-42, 42-46 and 46-50.

When the modulator domain is a nucleic acid, it typically modulates the activity of the cellular component by using base pairing as the predominant interaction. Further, the modulator domain may also mediate the synthesis of RNA and/or protein that modulates the activity of a cellular target. Hence in this embodiment, the modulator domain may be seen as an indirect modulator. Moreover, three dimensional structures may also be very important as is the case for aptamers.

The same applies to the situation where modulator domain is a polypeptide or a protein. Note, that as used herein, no distinction is made between a peptide, a polypeptide and a protein. As used herein, they are all characterised in being made up from amino acids. If a particular distinction is to be made with regards to e.g. a peptide, this will be explicitly noted. The modulator domain - polypeptides

In a preferred embodiment, modulator domain is a polypeptide selected from the group consisting of: an antibody, an enzyme, a peptide binding to a cellular target and a toxin.

As will be apparent to the skilled man, such polypeptides can all modulate the activity of a cellular target. Antibodies are particular preferred, as these can be developed with binding activity against most, if not all, cellular proteins. Techniques for generating high affinity monoclonal antibodies are well known to the skilled man. Even processes for generating human monoclonal antibodies are accessible to the skilled man. Human antibodies are particular preferred, because they are less immunogenic.

A peptide binding a cellular target can be identified by e.g. phage display. Also mRNA display, ribosome display, covalent display and other in vitro evolution methods can be used to generate high affinity peptides.

When the modulator domain is a peptide, is it preferred that the length of the peptide is selected from the group consisting of 6-8 amino acid residues, 8-10 amino acid residues, 10-12 amino acid residues, 12-14 amino acid residues, 14- 16 amino acid residues, 16-18 amino acid residues, 18-20 amino acid residues and 20-30 amino acid residues.

Peptidomimetic compounds may also be used for the modulator domain. Peptidomimetics will also be referred to below.

In a preferred embodiment, the modulator domain has a molecular weight of more than 1000 Dalton.

In other preferred embodiments, the modulator domain has a molecular weight from the group co more than consisting of: more than 2000 Dalton, more than 3000 Dalton, more than 4000 Dalton, more than 5000 Dalton, more than 6000 Dalton, more than 7000 Dalton, more than 8000 Dalton, more than 9000 Dalton, and more than 10000 Dalton. In another preferred embodiment, the cellular component is selected from the group consisting of: an mRNA, a miRNA, a tRNA, an rRNA, a transcription factor, a kinase and a polymerase.

Typically, the particular cellular component will be dependent on the modulator domain, as the cellular component and the modulator domain should interact such that the modulator domain can modulate the effect of the cellular component. I.e. the modulator is chosen such as to target a given cellular component. As outlined above, in some embodiments, the modulator domain may act as an indirect modulator. However, obviously the cellular component will still be dependent on the modulator domain.

In one embodiment, the modulator domain may be bound by a modulator binding domain, which is in turn covalently coupled to the lipophilic domain and the cationic domain, in analogy to the above discussion relating to modulator domains of nucleic acids. The modulator binding domain may e.g. be an antibody that binds the modulator domain or vice versa.

The cationic domain Turning now to the cationic domain, in preferred embodiments, the cationic domain is characterised in having a charge selected from the group consisting of: a charge of at least 4 positive charges at a pH of 7, a charge of at least 5 positive charges at a pH of 7, a charge of at least 6 positive charges at a pH of 7, a charge of at least 4 positive charges at a pH of 8, a charge of at least 9 positive charges at a pH of 7.

In an even more preferred embodiment, the cationic domain has a charge of at least 4 positive charges at a pH of 7.

Not intended to be bound by theory, a positive charge is important as it seems to be a characteristic of compounds that facilitate transport across a membrane.

Particular well-described cationic compounds that have these properties are peptides. Thus, in a preferred embodiment, cationic domain is a cationic peptide. Even more preferred is that the cationic peptide is a so called cell penetrating peptide. Examples of such cell penetrating peptides are the drosophila transcription factor pant (penetratin), pAnt, herpes simplex virus type-1 transcription factor V22, Tat peptide, Tatp from HIV-I transactivator, Tat, polyarginine and the galanin/mastoparan chimera and Transportan.

Preferably the cationic peptide comprises between 4 amino acid and 12 amino acid residues, at least 4 of which are independently selected from lysine and arginine residues.

The cationic domain may also be a peptide derivative, or a peptidomimetic. Hence, the cationic domain may e.g. be a peptoid (N-substituted glycine), a beta- peptide, a gamma peptide or combination thereof. The peptide may also comprise D-amino acids or even be fully build from D-amino acids. Peptidomimetics have various advantages, in particular with regards to bioavailability and biostability. Obviously, also the modulator domain may be a peptide derivative or peptidomimetic.

The lipophilic domain

The lipophilic domain of the present invention is characterized in that it increases the transmembrane delivery of a modulator domain coupled to a cationic domain. A typical example is an antisense such as a PNA coupled to a cationic peptide, wherein the cationic peptide improves the transmembrane delivery of the antisense. Such compounds are well-known. However, we have discovered that the transmembrane delivery of such compounds can be improved by the further coupling of a lipophilic domain.

In a preferred embodiment, lipophilic domain comprises an alkyl chain with a length selected from the group consisting of: at least 4 C atoms, at least 5 C- atoms, at least 6 C-atoms, at least 7 C-atoms, at least 8 C-atoms, at least 9 C- atoms, at least 10 C-atoms, at least 11 C-atoms, at least 12 C-atoms, at least 13 C-atoms, at least 14 C-atoms, at least 15 C-atoms, at least 16 C-atoms, at least 17 C-atoms, at least 18 C-atoms, at least 19 C-atoms and at least 20 C-atoms. Alkyl chains are by their nature lipophilic and the longer the alkyl chain is, the more lipophilic will the three-domain compound be. However, it is not known whether the overall lipophilicity of the three-domain compound is the determining factor in improving transmembrane delivery. The determining factor in improving transmembrane delivery efficiency could also lie solely in the lipophilicity of the lipophilic domain.

In another embodiment, the lipophilic domain comprises an alkenyl chain with a length selected from the group consisting of: at least 4 C atoms, at least 5 C- atoms, at least 6 C-atoms, at least 7 C-atoms, at least 8 C-atoms, at least 9 C- atoms, at least 10 C-atoms, at least 11 C-atoms, at least 12 C-atoms, at least 13 C-atoms, at least 14 C-atoms, at least 15 C-atoms, at least 16 C-atoms, at least 17 C-atoms, at least 18 C-atoms, at least 19 C-atoms and at least 20 C-atoms.

In still another embodiment, the lipophilic domain comprises an alkynyl chain with a length selected from the group consisting of: at least 4 C atoms, at least 5 C- atoms, at least 6 C-atoms, at least 7 C-atoms, at least 8 C-atoms, at least 9 C- atoms, at least 10 C-atoms, at least 11 C-atoms, at least 12 C-atoms, at least 13 C-atoms, at least 14 C-atoms, at least 15 C-atoms, at least 16 C-atoms, at least 17 C-atoms, at least 18 C-atoms, at least 19 C-atoms and at least 20 C-atoms.

When referring to the length of alkyl, alkenyl, and alkynyl chains, what is meant is the length of the longest linear alkyl chain. The chain may also comprise branches. Further, the lipophilic domain may comprise cycloalkyls and heterocycles that in turn may be polycyclic systems such as bi-cyclic, tri-cyclic and tetra-cyclic systems. The polycyclic systems may further be spiro ring systems, fused ring systems or fused ring systems.

The lipophilic domain may also comprise one or more aromatic rings.

Heteroaromatic rings may also be comprised in the lipophilic domain. Thus, in one embodiment, the lipophilic domain comprises at least 1 aromatic ring, such as at least 2 aromatic rings, such as at least 3 aromatic rings and such as at least 4 aromatic rings. The lipophilic domain may also comprise steroids such as cholesterol, cholic acid, or cortisone. Terpenes such as e.g. camphor may also be comprised within the lipophilic domain. Also adamantane (tricyclo[3.3.1.13,7]decane) or derivatives thereof may be comprised within the lipophilic domain.

In a very preferred embodiment, the lipophilic domain is a fatty acid that has been conjugated to either the modulator domain or to the cationic domain.

As used herein, the term fatty acid refers to a carboxylic acid with an aliphatic chain. Specific examples of fatty acids are described below.

Conjugation may be done in any suitable ways such as to covalently link the fatty acid to the modulator domain or the cationic domain. If the modulator domain or the cationic domain is a peptide, a convenient coupling method is coupling to an amino group, e.g. the side chain amino group of a lysine. Such coupling is outlined in the examples section. Various methods for coupling amino groups and carboxylic acids are known to the skilled man. Coupling can e.g. also be achieved using EDC (l-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and NHS (N-hydroxysulfosuccinimide).

In a preferred embodiment, the three-domain compound further comprises a cleavable linker. Preferably, the linker is not cleaved before the compound has entered a cell by transmembrane delivery. Once inside the cell, the linker may be cleaved e.g. because of acid lability or by enzymatic cleavage. The use of such a linker can have various advantages. Thus, releasing the modulator domain from the cationic domain and the lipophilic domain may enhance the activity of the modulator domain. In a particular preferred embodiment, the modulator is inactive when coupled to the cationic domain and the lipophilic domain, and activated when released from the cationic domain. This principle is particular interesting with regards to delivering a toxic payload to a cell. Typically, the toxin should only exert its effects inside target cells, and the target cells may be defined by a targeting moiety that directs the compound to certain cells. Such approaches have been described for treatment of cancer, except that the use of a lipophilic group to further improve transmembrane delivery has not been suggested. Thus, in a preferred embodiment, the modulator domain is linked to the cationic domain and the lipophilic domain by a cleavable linker.

Consequently, cleavage of the linker releases the modulator domain from the cationic domain and/or the lipophilic domain.

If the cleavable linker is acid labile, it will be cleaved if the three-domain compound enters lysosomes.

The cleavable linker may also be an enzyme cleavable linker. One such example is the tetrapeptide Gly-Phe-Leu-Gly, which is cleaved by lysosomal enzymes such as cathepsin B.

The linker may also be hydrolysable and selected from the group consisting of: - HNCO-, -CONH-, -COO-, -00C-, -NHCOO-, -OOCNH-, -NHCONH-, -S02NH-, - NHS02- and -0-.

When the lipophilic domain of the three-domain compound is a fatty acid, it is preferred that the fatty acid comprises a number of C-atoms selected from the group of: at least 4 C atoms, at least 5 C-atoms, at least 6 C-atoms, at least 7 C- atoms, at least 8 C-atoms, at least 9 C-atoms, at least 10 C-atoms, at least 11 C- atoms, at least 12 C-atoms, at least 13 C-atoms, at least 14 C-atoms, at least 15

C-atoms, at least 16 C-atoms, at least 17 C-atoms, at least 18 C-atoms, at least

19 C-atoms and at least 20 C-atoms

In another preferred embodiment, the fatty acid is selected from the group consisting of: butanoic acid; hexanoic acid; octanoic acid; decanoic acid; dodecanoic acid; tetradecanoic acid; hexadecanoic acid; 9-hexadecenoic acid; octadecanoic acid; 9- octadecenoic acid; 11-octadecenoic acid; 9,12-octadecadienoic acid; 9,12,15- octadecatrienoic acid; 6,9,12-octadecatrienoic acid; eicosanoic acid; 9-eicosenoic acid; 5,8,11,14-eicosatetraenoic acid; 5,8,11,14,17-eicosapentaenoic acid; docosanoic acid; 13-docosenoic acid; 4,7,10,13,16,19-docosahexaenoic acid and tetracosanoic acid. A most preferred fatty acid is decanoic acid.

As will be apparent from the examples section, some fatty acids are more effective than others in improving transmembrane delivery. Thus, if a cationic peptide very effective in transmembrane delivery is used, is may be desired not to use the most efficient fatty acid. There seems to be a fine balance between efficiency of delivery and toxicity of the three-domain compound, as will be apparent from the experimental section. Thus, it will most likely be not be possible to define the best lipophilic domain in general, as the best lipophilic domain will vary with the modulator domain and the cationic domain.

In a preferred embodiment, the cationic domain is a peptide and lipophilic domain is a fatty acid.

In a further preferred embodiment, the cationic domain is a peptide and lipophilic domain is a fatty acid is conjugated to the peptide at a lysine residue.

The following is an exemplary and non-exhaustive list of three-domain compounds that has been prepared and tested:

Tat-Lys(Deca)-Gly- CCT CTT ACC TCA GTT ACA -NH2 NLS-Lys(Deca)-Gly- CCT CTT ACC TCA GTT ACA -NH2 H-(Lys)6-Lys(Deca)-Gly- CCT CTT ACC TCA GTT ACA -NH2 H-(Lys)9-Lys(Deca)-Gly- CCT CTT ACC TCA GTT ACA -NH2 H-GRK KRR QRR R-Lys(Deca)-Gly- CCT CTT ACC TCA GTT ACA -NH2 H-(Arg)6-Lys(Deca)-Flk- CCT CTT ACC TCA GTT ACA-NH2 H-(Arg)6-Lys(0cta)- CCT CTT ACC TCA GTT ACA-NH2 H-(Arg)6-Lys(Dodeca)- CCT CTT ACC TCA GTT ACA-NH2 H-(Arg)6-Lys(Hexadeca)- CCT CTT ACC TCA GTT ACA-NH2 H-(D-Arg)6-Lys(Deca)-Gly~CCT CTT ACC TCA GTT ACA-NH2 H-(D-Arg)9-Lys(Deca)-Gly~CCT CTT ACC TCA GTT ACA-NH2 H-(Arg)6-Lys(Deca)-Gly-CCT CTG ACC TCA TTT ACA-NH2 H-(Arg)9-Lys(Deca)-Gly-CCT CTG ACC TCA TTT ACA-NH2 Tat-Lys(Deca)-Gly-CCT CTG ACC TCA TTT ACA-NH2 H- (D-Arg)7-Lys(Deca)- CCT CTT ACC TCA GTT ACA-NH2 H- (D-Arg)8-Lys(Deca)- CCT CTT ACC TCA GTT ACA-NH2

Deca- (D-Arg)7-Gly- CCT CTT ACC TCA GTT ACA-NH2

Deca- (D-Arg)δ-Gly-CCT CTT ACC TCA GTT ACA-NH2

H- (D-Arg)7-l_ys(Deca)-Gly-CCT CTT ACC TCA GTT ACA-NH2 H- (D-Arg)8-Lys(Deca)-Gly-CCT CTT ACC TCA GTT ACA-NH2

H-Lys(Deca)GR KKR RQR RRP PQLys(Deca) GCCT CTT ACC TCA GTT ACA-

NH2

H-l_ys(Deca)-(D-Arg D-Lys)3 GIy- CCT CTT ACC TCA GTT ACA-NH2

H-l_ys(Deca)-(D-Arg D-Lys)4 GIy- CCT CTT ACC TCA GTT ACA-NH2 H-Lys(Deca) (D-Arg D-Arg Ala)3 GIy- CCT CTT ACC TCA GTT ACA-NH2

H-Lys(Deca) (D-Arg D-Arg Ala)4 GIy- CCT CTT ACC TCA GTT ACA-NH2

H-Lys(Deca) (D-Arg)4(D-l_ys)4 GIy- CCT CTT ACC TCA GTT ACA-NH2

H-Lys(Deca) (D-l_ys)2(D-Arg)4(D-l_ys)2 GIy- CCT CTT ACC TCA GTT ACA-

NH2

-wherein K is lysine, R is arginine, F is phenylalanine, G is glycine, Q is glutamine, P is proline, V is valine, Tat is GRKKRRQRRRPPQ, NIs is PKKKRKV, Lys(Deca) is Nε-decanoyl lysine, Lys(Octa) is Nε-octanoyl lysine, Lys(Dodeca) is Nε-dodecanoyl lysine, Lys(Hexadeca) is Nε-hexadecanoyl lysine and wherein the nucleotide sequence is PNA.

Uses and methods

A second aspect of the invention is use of the three-domain compound for delivery of a biologically active agent across a biological membrane, wherein the biologically active agent is the modulator domain of the three-domain compound.

In a preferred embodiment of this second aspect, the biological membrane is the tight junctions of the blood brain barrier.

In another preferred embodiment of the second aspect, the biological membrane is a cellular membrane.

In another preferred embodiment, the delivery across a biological membrane occurs in vivo. In another preferred embodiment, the delivery across a biological membrane occurs ex vivo.

In another preferred embodiment, the delivery across a biological membrane occurs in vitro.

A third aspect of the invention is a method of enhancing delivery of a biologically active agent across a biological membrane, wherein the biological active agent is the modulator domain of the three-domain compound, and wherein the method comprises the steps:

a. Providing a cell b. Providing a three-domain compound c. Contacting the cell with the three-domain compound such as to allow transmembrane delivery.

The above method may be performed in vitro, ex vivo or in vivo.

Therapy As the three-domain compounds of the invention can be used to modulate the activity of cellular components, they are useful as medicaments.

Hence, a fourth aspect of the invention is use of the three-domain compound of the invention as a medicament.

Still another aspect, is use of the three-domain compound for the preparation of a medicament for the prevention or treatment of one or more diseases selected from the group consisting of diseases resulting from viral infections, cancer, cardiovascular diseases, autoimmune diseases, inflammatory diseases and respiratory diseases.

Still another aspect of the invention is a pharmaceutical composition comprising the three-domain compound and a physiologically acceptable carrier and/or a physiologically acceptable diluent. Still another aspect is a method of treatment or prevention of a disease, said method comprising administering a pharmaceutical composition comprising the three-domain compound to a subject in need thereof.

Yet another aspect of the invention is a commercial kit of parts comprising at the three-domain compound.

In a preferred embodiment, the kit further comprises one or more components selected from the group consisting of buffers, media, carriers, diluents, radioactive or non-radioactive labels, positive controls, cells to be transfected, and instructions for use.

A final aspect of the invention is a method of producing the three-domain compound, said method comprising the steps of:

a. providing a cationic domain b. providing a lipophilic domain c. providing the modulator domain d. coupling the cationic domain and the lipophilic domain to the modulator domain.

Figure legends

Fig. 1. Relative cellular uptake of the PNA conjugates in HeLa pLuc705 cells.

Fig. 2. Relative cellular uptake of the PNAs conjugated with cell penetrating peptides

Fig. 3. Comparison of the PNA conjugates for the cellular uptake in the HeLa pLuc705 cells.

Fig. 4. Comparison of the PNAs conjugated with a Lys(decanoic acid) moiety and a different length of oligo L-arginine or oligo D-arginine for the cellular uptake in the HeLa pLuc705 cells.

Fig. 5. Comparison of the PNAs conjugated with hexaarginine ((Arg)6-) and different length of aliphatic chain for the cellular uptake in the HeLa pLuc705 cells.

Fig. 6. Relative cellular uptake of the PNAs conjugated with a different cell penetrating peptide (CPP) or both CPP and Lys(Deca) moiety for the cellular uptake in the HeLa pLuc705 cells.

Fig. 7. Antisense splice correction effect of a cholesteryl-octaarginine PNA conjugate.

Fig. 8. A PNA-Catϋp conjugate in which the PNA is partially complementary to the siRNA antisense strand is used to form the siRNA-Catϋp hybridization complex.

Fig. 9. Sequence of the anti luciferase antisense siRNA and the PNA-Catϋp conjugates used for delivery.

Fig. 10. Antisense strand siRNA delivery by CatLip-PNAs.

Fig. 11. CatLip peptides used for cellular delivery of anti-luciferase siRNA to p53R cells.

Fig. 12. Comparison of the efficiency of different length CatLip peptides for delivery of duplex siRNA.

Fig. 13. Effect of fatty acid length of CatLip peptide for on siRNA delivery. Fig. 14. Comparison of CatLip peptides and LFA2000 for siRNA delivery.

Examples

PNA synthesis

The PNAs were synthesized on a Boc-A-4-methylbenzhydrylamine resin (loading 5 0.12 mmol/g) using the standard synthetic protocol (1). The fatty acids were attached to the ε-amino group of a lysine. This lysine was incorporated using ε - fmoc protection, and the fmoc group was removed by standard piperidine treatment. Then the fatty acid was coupled using standard coupling conditions (1), and the synthesis was continued on the support with the peptide part. In 10 some cases the peptide was synthesized before attachment of the fatty acid via an N-terminal lysine.

The resulting PNA-conjugates were deprotected and cleaved from the resin with a cocktail composed of m-cresol/thioanisole/trifluoromethanesulfonic acid/TFA

15 (1/1/2/6, v/v) and the crude material was precipitated and washed with anhydrous (crucial to ensure ester stability) diethyl ether. The PNA conjugates were then purified by reversed -phase high performance liquid chromatography (HPLC) using a C18 column and an acetonitrile gradient in 0.1% TFA in H2O, and characterized by MALDI-TOF mass spectroscopy.

20

Alternatively, a peptide fatty acid conjugate also containing a cysteine was synthesized and purified as described above, and this was conjugated to a maleimide derivatized PNA as described in (Koppelhus L), Awasthi SK, Zachar V, Hoist HL), Ebbesen P, Nielsen PE: Cell-dependent differential cellular uptake of

25 PNA, peptides, and PNA-peptide conjugates. Antisense & Nucleic Acid Drug Development 2002, 12: 51-63.)

Cellular uptake experiments using the "positive read-out" reporter cell line, pLuc705 HeLa

The pLuc705 HeLa cell line was purchased from Gene Tools (Oregon, USA). pLuc 30 705 HeLa cells are derived by stable integration of the pLuc705 plasmid into HeLa S2 cells. The plasmid has luciferase downstream and out-of-frame with a dominant splice-mutation in the beta-globin intron position 705. The recombinant luciferase gene is under control of the immediate early cytomegalovirus promoter. Binding of PNA (of sequence H-CCT CTT ACC TCA GTT ACA -NH2) to a region covering the splice mutation results in restoration of wildtype splicing which generates a transcript with luciferase in-frame. pLuc 705 HeLa cells were grown in Dulbecco ^'s modified Eagle ^'s medium (DMEM) supplemented with penicillin/streptomycin (10 μg/mL) (antibiotics) and 10 % fetal calf serum (Gibco, Invitrogen, Denmark). Cell cultures were maintained at 370 C in a humidified atmosphere containing 5 % CO₂ (standard conditions). For transfection with the PNA conjugates, the cells were seeded in a 96 well microtiterplate (Nunc, Invitrogen, Denmark) in a volume of 200 μl_ and at cell densities of 50-100 x 10³ cells/ml unless otherwise stated. The following day the semi-confluent cells were prepared for uptake experiments. This was done by washing the cells once in fresh DMEM containing antibiotics but no serum (serum- free DMEM) and adding 80 -90 μl of the serum-free DMEM (unless otherwise stated) to the cells afterwards. The cells were then incubated at standard conditions for 1-2 hours before the cells were given CatLip-PNA conjugates in the concentrations mentioned in the text. Typically, the cells were given 2-10 μl_ of a lOOμM stock. All additions were made in at least triplicates. Control cells were given sterilized water in volumes similar to each different volume of added CPP- PNA stock. This was done to avoid any misinterpretations due to volume effects. After typically 4 hours all cultures were added lOμL fetal calf serum. After incubation at standard conditions for 16-20 hours, luciferase activity was measured by use of the Bright-GloTM Luciferase Assay System (Promega, Ramcon A/S, Birkerød, Denmark) and a Victor2 Multilabel Counter (Wallac, Perkin-Elmer Danmark A/S, Allerød, Denmark) according to manufacturers instructions.

Cell viability was assessed by use of a MTS assay (Promega) or by indirect ATP measurements performed on cells run in parallel and treated exactly as the cells used for Luciferase determinations. ATP was determined by use of the ATPIite assay (Perkin-Elmer Danmark A/S, Allerød, Denmark) according to manufactures instructions

RT-PCR

Transfection experiments were performed as described above but scaled up to be performed in 24 well titer plates. RNA was isolated by use of the RNeasy Mini kit (Qiagen, Albertslund, Denmark) according to manufacturer's instruction. Two nanograms of total RNA were used for each RT-PCR reaction (10 μl). RT PCR was performed by using OneStep RT-PCR kit (Qiagen) according to manufacturer's instruction: Primers for RT-PCR were as follows: forward primer, 5'- TTGATATGTGGATTTCGAGTCGTC-S'; reverse primer, 5'-TGTCAAT- CAGAGTGCTTTTGGCG-3'. RT-PCR program was as follows: [(55⁰C, 35 min), 1 cycle; (95⁰C, 15 min), 1 cycle; (94⁰C, 0.5 min; 55⁰C, 0.5 min; 72⁰C, 0.5 min), 29 cycles]. RT-PCR products were analyzed on 2% agarose gel run in Ix TBE buffer and visualized by ethidium bromide staining. Gel images were captured by ImageMaster (Amersham Biosciences, Hillerød, Denmark) and analyzed by L)N- SCAN-IT software (Silk Scientific Corporation, USA).

Example 1

Relative cellular uptake of the PNA conjugates in HeLa pLuc705 cells.

The text refers to fig. 1.

Cells, plated in the 96 well plate, were transfected with PNAs(Tat-PNA, Deca-PNA, Tat-Deca-PNA and Deca-Tat-Deca-PNA) for 4 h in the OPTI-MEM medium at the designated concentrations (0 - 8 μM) and incubated further for 24 h after addition of the serum containing growth medium without removing the transfection solution. Then cells were subjected to the further analysis. All tests were performed in triplicate and the results are given as the average values ± standard deviations (SD). (A) Luciferase activities were analyzed by Blight-Gro reagent (Promega) and shown as relative light units (RLU/well). (B) Cellular viabilities were analyzed by MTS-assay (Promega) and obtained values were normalized to the average value of non-treated control.

Example 2

Relative cellular uptake of the PNAs conjugated with cell penetrating peptides.

The text refers to fig. 2.

Relative cellular uptake of the PNAs conjugated with cell penetrating peptides ((D- Arg)₇-, (D-Arg)₈- or Tat-) and/or Lys (Decanoic acid) moiety for the cellular uptake in the HeLa pLuc705 cells. Cells, plated in 96 well plate, were transfected with PNAs (0-8 μM) for 4 h in the OPTI-MEM medium and incubated further for 24 h after addition of the serum containing growth medium without removing the transfection solution. Then cells were subjected to the luciferase analysis. Luciferase activities were analyzed by Blight-Gro reagent (Promega) and shown as relative light units (RLU/well). All tests were performed in triplicate and the results are given as the average values ± standard deviations (SD).

Example 3

Comparison of the PNA conjugates for the cellular uptake in the HeLa pLuc705 cells.

The text refers to fig. 3.

Cells, plated in 24 well plate, were transfected with PNAs for 4 h in the OPTI-MEM medium and incubated further for 24 h after addition of the serum containing growth medium. Then cells were subjected to the further analysis. (A) PNAs (Deca-Tat-Deca-PNA, Tat-Deca-PNA, (D-Arg)₇-Deca-PNA, (D-Arg)₈-Deca-PNA and (D-Arg)₈-PNA were transfected to the cells at the designated concentrations (0-6 μM). (3A) Luciferase activity were measured by Luciferase assay system (Promega) and normalized to protein concentrations (shown as RLL)/ μg of protein). (3B) RT-PCR analysis of the mis-splicing correction of pre-mRNAs by PNAs. Total RNAs were extracted from the cells after transfection and subjected to the RT-PCR analysis. Uncorrected indicates the 268 bp fragment without mis- splicing correction, and corrected indicated the 142 bp fragment with mis-splicing correction. Numbers under each lane indicated the relative amount of the corrected form to the sum of corrected form and uncorrected form. (B) Tat-Deca- PNA and its mismatch PNA were transfected to the cells at the designated concentrations (0-8 μM). (3D) Luciferase activity were measured by Luciferase assay system (Promega) and normalized to protein concentrations (shown as RLL)/ μg of protein). (3C) RT-PCR analysis of mis-splicing correction by PNAs. Analysis was performed as described in (3B).

Example 4

Comparison of the PNAs conjugated with a Lys(decanoic acid) moiety and a different length of oligo L-arginine or oligo D-arginine for the cellular uptake in the HeLa pLuc705 cells. The text refers to fig. 4.

Cells, plated in 96 well plate, were transfected with PNAs (R6, (Arg)6-Deca-PNA; R7, (Arg)₇-Deca-PNA; R8, (Arg)₈-Deca-PNA; R9, (Arg)g-Deca-PNA); r6, (D-Arg)₆- Deca-PNA; r7, (D-Arg)₇-Deca-PNA; r8, (D-Arg)₈-Deca-PNA and r9, (D-Arg)g-Deca- PNA) for 4 h in the OPTI-MEM medium at the designated concentrations (0-8 μM) and incubated further for 24 h after addition of the serum containing growth medium. Then cells were subjected to the further analysis. All tests were performed in triplicate and the results are given as the average values ± standard deviations (SD). (A) Luciferase activities were analyzed by Blight-Gro reagent (Promega) and shown as relative light units (RLU/well). (B) Cellular viabilities were analyzed by MTS-assay (Promega) and obtained values were normalized to the average value of non-treated control.

Example 5

Comparison of the PNAs conjugated with hexaarginine ((Arg)₆-) and different length of aliphatic chain for the cellular uptake in the HeLa pLuc705 cells

The text refers to fig. 5.

Cells, plated in the 96 well plate, were transfected with PNAs ((Arg)₆-Octa-PNA, (Arg)₆-Deca-PNA, (Arg)₆-Dodeca-PNA and (Arg)₆-Hexadeca-PNA) for 4 h in the OPTI-MEM medium at the designated concentrations (0-4 μM) and incubated further for 24 h after addition of the serum containing growth medium without removing transfection solution. Then cells were subjected the further analysis. All tests were performed in triplicate and the results are given as the average values ± standard deviations (SD). (A) Luciferase activities were analyzed by Blight-Gro reagent (Promega) and shown as relative light units (RLU/well). (B) Cellular viabilities were analyzed by MTS-assay (Promega) and obtained values were normalized to the average value of non-treated control. Example 6

Relative cellular uptake of the PNAs conjugated with a different cell penetrating peptide (CPP) or both CPP and Lys(Deca) moiety for the cellular uptake in the HeLa pLuc705 cells.

The text refers to fig. 6.

Cells, plated in the 96 well plate, were transfected with PNAs ((RK)₃-PNA, Deca- (RK)₃-PNA, (RK)₄-PNA, DeCa-(RK)₄-PNA, (RRA)₃-PNA, DeCa-(RRA)₃-PNA, (RRA)₄- PNA, DeCa(RRA)₄-PNA, R₄K₄-PNA, DeCa-R₄K₄-PNA, K₂R₄K₂-PNA, DeCa-K₂ R₄K₂-PNA, Tat-PNA, Tat-Deca-PNA, (Arg)₉-PNA, (D-Arg)g-Deca-PNA) for 4 h in the OPTI-MEM medium at the designated concentrations (0-4 μM) and incubated further for 24 h after addition of the serum containing growth medium. Then cells were subjected to the luciferase analysis. Luciferase activities were analyzed by Blight-Gro reagent (Promega) and shown as relative light units (RLU/well). All tests were performed in triplicate and the results are given as the average values ± standard deviations (SD).

Example 7 Antisense splice correction effect of a cholesteryl-octaarginine PNA conjugate in the HeLa cell luciferase assay compared to that of the Tat-Deca-PNA. It is observed that the cholesteryl-octaarginine PNA conjugate is considerable more active than the Tat-Deca-PNA (2 mM) but also more toxic (6 mM), and the effect of the cholesteryl-octaarginine PNA is not enhanced by chloroquine. The conjugate was synthesized by EDC activated N-terminal conjugation of cholesteryl succinic ester with the octaarginine PNA.

Results & Discussion of examples 1-7

The Tat peptide belong to the group of cell penetrating peptides that was originally introduced for cellular delivery and for which it has subsequently been shown that cellular uptake is quite efficient. We chose to prepare antisense PNA-Tat-fatty acid conjugates targeted to the aberrant splice site in the engineered luciferase gene in pLucHeLa cells. This antisense assay yields a positive readout (luciferase activation) that is readily quantified with high sensitivity, and therefore allows reliable and quantitative comparison of different delivery methods (for antisense agents). The results presented in Figure 1 clearly demonstrate that while the PNA-Tat conjugate shows only very weak antisense activity (at 8 μM), the analogous conjugate containing a decanoic acid attached to the ε-amino group of a lysine (Tat-Deca-PNA) is at least ten times more active, and the activity is further enhanced up to 5 fold (compare activities at 2 μM) by having two decanoic acids in the conjugate (Deca-Tat-Deca- PNA). However, increased cellular toxicity is observed for the Tat-fatty acid conjugates and this is especially pronounced for the di-fatty acid compound. Indeed, the antisense activity of this is reduced at higher concentrations (4 & 8 μM), and this is ascribed to cellular toxicity. The control fatty acid-PNA conjugate (Deca-PNA) showed no activity.

In order to address whether conjugation of a lipophilic moiety (a fatty acid) to a cationic cell penetrating peptide domain could be a general principle to obtain increased cellular bioavailability we made a series of PNA conjugates based on oligo-arginines. The results presented in Figure 2 show that although hepta- and octa-arginine PNA conjugates as reported previously show significantly higher antisense activity than the PNA-Tat conjugate, the activity is further enhanced 2-3 fold by fatty acid conjugation. The effect is observed up to 4 μM but is not seen at 8 μM due to increased toxicity of the fatty acid conjugates. We also note that although the Tat-Deca-PNA conjugate is slightly less active than the Arg-Deca- PNA conjugates at lower concentrations, it exhibits the highest activity at 8 μM because of lower toxicity. The results also show that the position of the fatty acid relative to the peptide (amino terminal (e.g. Deca-(D-Arg)₇-PNA) or C-terminal between the peptide and the PNA (e.g. (D-Arg)7-Deca-PNA) does not seem of major importance for activity.

The luciferase activity reading is a secondary effect from the molecular event of splicing redirection by the PNA, and we therefore found it important to verify the antisense effect directly at the mRNA level. The results show a very good correlation between the luciferase activity (Figure 3) and the relative amount of correctly spliced luciferase mRNA at PNA concentrations up to 4 μM. However, at 6 μM where toxicity becomes a limiting factor and where the luciferase readings are reduced for two of the conjugates (PNAs 2821 & 2784), the RNA data shows increased relative correction activity also for these two PNAs, indicating an expected dose response on splice correction in spite of a decreased amount of total mRNA due to general toxicity. As a control for the sequence specificity of the antisense effect we synthesized a double base pair mismatch derivative of identical base composition (two base interchange) of the Tat-Deca-PNA and tested this. As expected, this PNA exhibited significantly reduced activity both in terms of luciferase activity as well as in terms of mRNA correction activity.

A comparison between D-Arg and L-Arg conjugates (Figure 4) shows slightly higher activity of the non-natural D-form clearly arguing against the involvement of specific receptor interactions of the peptide. These data also stress the very fine balance between uptake and toxicity.

At 2 μM the activity increases with increasing number of arginines, but as the concentration increases this order changes as the longer oligo-arginines are relatively more toxic than the shorter ones. Finally, the D-Arg conjugates show higher antisense activity but in parallel are also more toxic to the cells.

This delicate activity/toxicity balance is further illustrated when analysing the effect of the length of the fatty acid. For this experiment we chose the less active and less toxic hexa-arginine conjugate (Figure 5). Clearly, at lower PNA concentrations (up to 2 μM) the conjugates with longer fatty acid tails are the more active, but as the dodeca- and hexadeca-conjugates are clearly more toxic than the deca-conjugate, the latter shows the highest activity at 4 μM. Thus increased fatty acid length, and thus lipophilicity, produces more potent antisense agents but also compounds of higher cellular toxicity, and decanoic acid appears a good compromise.

A small series of arginine rich peptides was tested for enhancement by fatty acid acylation as illustrated by the data presented in Figure 6. It is clear that for all conjugates the acylated form exhibit superior activity relative the non-acylated form, but it is also noteworthy that the relative enhancement differs for the various peptides as already demonstrated by the difference between the Tat and oligo-arginine conjugates. In particular, the very dramatic enhancement (up to two orders of magnitude) of the (Arg-Arg-Ala)4 conjugate upon fatty acid conjugation is interesting in terms of the molecular mechanism(s) of the cellular uptake as this conjugate resembles the behaviour of the Tat peptide more than that of the oligo-arginines. These data would indicate a clear possibility for optimizing the peptide-fatty acid (CatLip) moiety in terms of delivery/toxicity ratio.

Example 8

Cellular delivery of siRNA

Experimentals:

Antisense strand siRNA delivery by CatLip-PNAs as shown in figure 10: p53R cells (expressing luciferase) were transfected with asRNA (antisense siRNA strand) in combination with different PNAs in the absence or presence of the enhancer chloroquine (CQ) Antisense siRNA strand: cPNA-CPP=l : l. LFA2000 (2 μl/ml).

After 48 hrs luciferase activity was measured according to the manufacturers instructions. It is clearly observed that effeicient RNA interference is observed with CatLip R8-Deca-PNA (marked by upper arrow) in the presence of chloroquine. The activity is approaching that of Lipofectamine (LFA2000, marked by lower arrow) aided delivery.

Comparison of the efficiency of different length CatLip peptides for delivery of duplex siRNA as shown in figure 12: siRNA (double strand) was complexed with Cat-Lip for 30 min before addition to the p53R cells. 48 h transfection. Final concentration of Cat-Lip peptides was 0 -4 μM.

Studying the effect of fatty acid length of CatLip peptide for on siRNA delivery as shown in figure 13: siRNA (double strand) was complexed with Cat-Lip peptide for 30 min before addition to the p53R cells. 48 h transfection. Final cone, of Cat-Lips peptides was 0-2 μM. It is seen that the longer fatty acid CatLip peptides are more efficient. Comparison of CatLip peptides and LFA2000 for siRNA delivery as shown in figure 14:

The p53R cells were transfected with siRNA (40 nM), LFA2000 (2 μl/ml) and CatLip peptide (2 μM). Luciferase activity was measured 48 h after transfection. It is seen that Deca-R10 and Stearyl-R8 has activity not dramatically different from LFA2000.

Results and discussion:

Previously cell penetrating peptides have been used for cellular delivery of oligonucleotide antisense and RNA interference agents through chemical conjugation of the peptide to the oligonucleotide. Here we demonstrate that the modulator domain may be coupled to the cationic domain and the lipophilic domain via (reversible) base pairing, as outlined in the detailed description of the invention. This will allow the modulator domain, in this case an oligonucleotide RNA interference agent, to dissociate from the peptide once inside the cell.

The experiments of example 8 illustrate this novel principle. Using PNA -peptide conjugates in which the PNA shares 8 base pairs hybridization overlap with the antisense siRNA strand targeting a luciferase reporter gene (Figure 9).

The results (Figure 10) demonstrate that (when using chloroquine activation) the PNA-CatLip conjugate, R8-Deca-PNA, is able to mediate siRNA down regulation of the luciferase close to that achieved using state of the art lipofectamin (LFA2000) siRNA delivery. It is also very significant that using similar conditions the analogous PNA-CatLip conjugate, R8-PNA, not containing the lipophilic decanoic acid domain does not confer siRNA activity.

In a related strategy, CatLip constructs in which the RNA binding domain and the cell delivery domain both are contained in the cationic peptide (Figure 11) are also able to deliver active siRNAs to eukaryotic cells (Figures 12-14). In this case, the more active reagents are found having 8 to 9 arginines (Figure 12) and a longer fatty acid chain (palmitic acid) (Figure 13), and these agents exhibit siRNA delivery capabilities close to that of state of the art lipofectamin (LFA2000) (Figure 14). In an extension of this approach three domain delivery agents analogous to those presented in figure 11 but containing a alternative RNA binding domain (instead of the PNA sequence) such as an nucleic acid ϊntercalator (e.g. amϊnoacrϊdϊne) or a minor groove binder (Hoechst dye) or an RNA binding aminoglycoside are obvious possibilities.

Claims

1. A compound comprising :

2. A compound according to claim 1, wherein the modulator domain, the cationic domain and the lipophilic domain are three distinct domains.

3. A compound according to any of the preceding claims, wherein the modulator domain is selected from the group consisting of polypeptides, nucleic acids and derivatives and mimics thereof.

4. A compound according to claim 3, wherein the nucleic acid is selected from the group consisting of: an antisense molecule, a siRNA, a miRNA, a ribozyme, a triplex forming oligonucleotide, an aptamer, a DNA or a plasmid.

5. A compound according to claim 3, wherein the nucleic acid comprise DNA, RNA, LNA, PNA, morpholino or any combinations thereof.

6. A compound according to any of the preceding claims, wherein the modulator domain is a polypeptide selected from the group consisting of: an antibody, an enzyme, a peptide binding a cellular target and a toxin.

7. A compound according any of the preceding claims, wherein the modulator domain has a molecular weight of more than 1000 Da.

8. A compound according to any of the preceding claims, wherein the cellular component is selected from the group consisting of: an mRNA, a miRNA, a tRNA, an rRNA, a transcription factor, a kinase and a polymerase.

9. A compound according to any of the preceding claims, wherein the cationic domain has a charge selected from the group consisting of: a charge of at least 4 positive charges at a pH of 7, a charge of at least 5 positive charges at a pH of 7, a charge of at least 6 positive charges at a pH of 7, a charge of at least 4 positive charges at a pH of 8, a charge of at least 9 positive charges at a pH of 7.

10. A compound according to claim 9, wherein the cationic domain has a charge of at least 4 positive charges at a pH of 7.

11. A compound according to any of claims 9 and 10, wherein the cationic domain is a cationic peptide.

12. A compound according to claim 11, wherein the cationic peptide is a cell penetrating peptide.

13. A compound according to claim 12, wherein the cationic peptide comprises between 4 amino acid and 12 amino acid residues, at least 4 of which are independently selected from lysine and arginine residues.

14. A compound according to any of the preceding claims, wherein the lipophilic domain has a log(P) value that is the same or is larger than the log(P) value of 1-hexanol

15. A compound according to any of the preceding claims, wherein the lipophilic domain has a log(D) value that is the same or is larger than the log(D) value of hexanoic acid

16. A compound according to any of the preceding claims, wherein the lipophilic domain comprises an alkyl chain with a length selected from the group consisting of: at least 4 C atoms, at least 5 C-atoms, at least 6 C- atoms, at least 7 C-atoms, at least 8 C-atoms, at least 9 C-atoms, at least 10 C-atoms, at least 11 C-atoms, at least 12 C-atoms, at least 13 C-atoms, at least 14 C-atoms, at least 15 C-atoms, at least 16 C-atoms, at least 17 C-atoms, at least 18 C-atoms, at least 19 C-atoms and at least 20 C- atoms.

17. A compound according to any of the preceding, wherein the lipophilic domain comprises an alkenyl chain with a length selected from the group consisting of: at least 4 C atoms, at least 5 C-atoms, at least 6 C-atoms, at least 7 C-atoms, at least 8 C-atoms, at least 9 C-atoms, at least 10 C- atoms, at least 11 C-atoms, at least 12 C-atoms, at least 13 C-atoms, at least 14 C-atoms, at least 15 C-atoms, at least 16 C-atoms, at least 17 C- atoms, at least 18 C-atoms, at least 19 C-atoms and at least 20 C-atoms.

18. A compound according to any of the preceding claims, wherein the lipophilic domain comprises an alkynyl chain with a length selected from the group consisting of: at least 4 C atoms, at least 5 C-atoms, at least 6

C-atoms, at least 7 C-atoms, at least 8 C-atoms, at least 9 C-atoms, at least 10 C-atoms, at least 11 C-atoms, at least 12 C-atoms, at least 13 C- atoms, at least 14 C-atoms, at least 15 C-atoms, at least 16 C-atoms, at least 17 C-atoms, at least 18 C-atoms, at least 19 C-atoms and at least 20 C-atoms.

19. A compound according to any of the preceding claims, wherein the lipophilic domain comprises at least one cycloalkyl or heterocycle.

20. A compound according to any of the preceding claims, wherein the lipophilic domain comprises at least 1 aromatic ring

21. A compound according to claims any of the preceding claims, wherein the lipophilic domain comprise a fatty acid that has been conjugated to either the modulator domain or to the cationic domain.

22.A compound according to claims any of the preceding claims, wherein the lipophilic domain is a fatty acid that has been conjugated to either the modulator domain or to the cationic domain.

23.A compound according to any of claims 21 and 22, wherein the fatty acid is selected from the group consisting of:

butanoic acid; hexanoic acid; octanoic acid; decanoic acid; dodecanoic acid; tetradecanoic acid; hexadecanoic acid; 9- hexadecenoic acid; octadecanoic acid; 9-octadecenoic acid; 11- octadecenoic acid; 9,12-octadecadienoic acid; 9,12,15- octadecatrienoic acid; 6,9,12-octadecatrienoic acid; eicosanoic acid; 9-eicosenoic acid; 5,8,11,14-eicosatetraenoic acid; 5,8,11,14,17- eicosapentaenoic acid; docosanoic acid; 13-docosenoic acid;

4,7,10,13,16,19-docosahexaenoic acid and tetracosanoic acid.

24.A compound according to any of claims 21-22, wherein the fatty acid comprises a number of C-atoms selected from the group of: at least 4 C atoms, at least 5 C-atoms, at least 6 C-atoms, at least 7 C-atoms, at least 8 C-atoms, at least 9 C-atoms, at least 10 C-atoms, at least 11 C-atoms, at least 12 C-atoms, at least 13 C-atoms, at least 14 C-atoms, at least 15 C- atoms, at least 16 C-atoms, at least 17 C-atoms, at least 18 C-atoms, at least 19 C-atoms and at least 20 C-atoms

25. A compound according to any of the preceding claims further comprising a cleavable linker

26.A compound according to claim 25, wherein cleavage of the linker releases the modulator domain from the cationic domain and the lipophilic domain.

27.A compound according to any of claims 25 and 26, wherein the linker is acid labile for cleavage in lysosomes

28.A compound according to any of claims 25 and 26, wherein the linker is hydrolysable and selected from the group consisting of: -HNCO-, -CONH-, - COO-, -00C-, -NHCOO-, -OOCNH-, -NHCONH-, -S02NH-, -NHS02- and -O-

29.A compound according to any of claims 25 and 26, wherein the linker is a enzyme cleavable linker such as e.g. the tetrapeptide Gly-Phe-Leu-Gly, which is cleaved by lysosomal enzymes such as cathepsin B

30. A compound according to any of the previous claims, wherein the cationic domain is a peptide and the fatty acid is conjugated to the peptide at a lysine residue

31. Use of a compound according to any of the preceding claims as a medicament.

32. Use of a compound according to any of claims 1-30 for the preparation of a medicament for the prevention or treatment of one or more diseases selected from the group consisting of diseases resulting from viral infections, cancer, cardiovascular diseases, autoimmune diseases, inflammatory diseases and respiratory diseases.

33. Use of a compound according to any of claims 1-30 for delivery of a biologically active agent across a biological membrane

34. Use according to claim 33, wherein the biological membrane is the tight junctions of the blood brain barrier.

35. Use according to claim 34, wherein the biological membrane is a cellular membrane.

36. Use according to any of claims 33-35, wherein transport of said one or more biologically active agents across said biological membrane occurs in vivo.

37. Use according to any of claims 33-35, wherein transport of said one or more biologically active agents across said biological membrane occurs in vitro.

38.A method of enhancing delivery of one or more biological agents across a biological membrane, said method comprising contacting a biological membrane with a three-domain compound according to any of claims 1- 30, wherein the biological agent is the modulator domain of the compound.

39.A pharmaceutical composition comprising the compound according to any of claims 1-30 and a physiologically acceptable carrier and/or a physiologically acceptable diluent.

40. A method of treatment or prevention of a disease, said method comprising administering to a subject in need thereof a therapeutically effective amount of a composition according to claim 39.

41. A commercial kit of parts comprising at least one compound according to any of claims 1 - 30.

42.A kit according to claim 41 further comprising one or more components selected from the group consisting of buffers, media, carriers, diluents, radioactive or non-radioactive labels, positive controls, cells to be transfected, and instructions for use.

43.A method of preparing the compound of any of claims 1-30, comprising the steps of:

a. providing a cationic domain b. providing a lipopilic domain c. providing a modulator domain d. coupling the cationic domain and the lipopilic domain to the biologically active agent