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WO2008043366A2 - Composés à trois domaines pour une administration transmembranaire - Google Patents

Composés à trois domaines pour une administration transmembranaire Download PDF

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Publication number
WO2008043366A2
WO2008043366A2 PCT/DK2007/050149 DK2007050149W WO2008043366A2 WO 2008043366 A2 WO2008043366 A2 WO 2008043366A2 DK 2007050149 W DK2007050149 W DK 2007050149W WO 2008043366 A2 WO2008043366 A2 WO 2008043366A2
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atoms
domain
acid
compound according
cationic
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WO2008043366A3 (fr
Inventor
Uffe Koppelhus
Peter Eigil Nielsen
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Københavns Universitet
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Københavns Universitet
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers

Definitions

  • a molecule e.g. a therapeutic agent
  • a molecule e.g. a therapeutic agent
  • 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.
  • CPP cell-penetrating peptides
  • 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.
  • 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.
  • a lipophilic domain such as a fatty acid.
  • 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.
  • a first aspect of the present invention is a compound comprising :
  • a modulator domain capable of modulating the activity of a cellular component
  • the lipophilic domain increases the transmembrane delivery of the modulator domain coupled the cationic domain.
  • a domain refers to a particular part of the compound that is structurally and/or functionally distinct from the other parts of the compound.
  • 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.
  • 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.
  • the lipophilic domain is characteristic in that it is more lipophilic than 1-hexanol.
  • the lipophilic domain is characteristic in that it is more lipophilic than hexanoic acid.
  • the lipophilicity of the lipophilic domain 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 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.
  • 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 lipophilic domain has a log(P) value that is that same or is larger than the log(P) value of 1-hexanol.
  • 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.
  • the lipophilic domain decreases the solubility of the three-domain compound in water, as compared to the same compound without the lipophilic domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the three-domains together make up more than 80% of the total molecular weight of the compound.
  • 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 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.
  • nucleic acids can modulate the activity of a cellular target.
  • these nucleic acids may be delivered with a complementary counterpart that is not covalently coupled to the compound.
  • 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.
  • 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.
  • a nucleic acid serving as a modulator binding domain
  • the modulator domain is covalently coupled to the lipohilic domain and the cationic domain.
  • 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.
  • a microRNA, aptamer etc. may be base paired to a nucleic acid that is covalently coupled to the lipophilic domain and the cationic domain.
  • the region of base pairing does not interfere with the activity of the modulator domain (microRNA, aptamer, siRNA etc. ).
  • 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.
  • nucleic acid and its analogues and mimics are DNA, RNA, LNA, PNA, morpholino or any combinations thereof.
  • nucleotide analogues/mimics capable of sequence specific base pairing may also be comprised within the nucleic acid.
  • 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.
  • the modulator domain 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.
  • modulator domain is a polypeptide or a protein.
  • a peptide a polypeptide and a protein.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • the modulator domain has a molecular weight of more than 1000 Dalton.
  • 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.
  • 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.
  • 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.
  • the modulator is chosen such as to target a given cellular component.
  • the modulator domain may act as an indirect modulator. However, obviously the cellular component will still be dependent on the modulator domain.
  • 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 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.
  • the cationic domain has a charge of at least 4 positive charges at a pH of 7.
  • a positive charge is important as it seems to be a characteristic of compounds that facilitate transport across a membrane.
  • cationic domain is a cationic peptide.
  • the cationic peptide is a so called cell penetrating peptide.
  • 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.
  • 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.
  • 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.
  • the modulator domain may be a peptide derivative or peptidomimetic.
  • 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.
  • 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.
  • 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.
  • 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.
  • alkyl, alkenyl, and alkynyl chains 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.
  • 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.
  • 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.
  • adamantane (tricyclo[3.3.1.13,7]decane) or derivatives thereof may be comprised within the lipophilic domain.
  • the lipophilic domain is a fatty acid that has been conjugated to either the modulator domain or to the cationic domain.
  • 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.
  • 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).
  • the three-domain compound further comprises a cleavable linker.
  • 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.
  • releasing the modulator domain from the cationic domain and the lipophilic domain may enhance the activity of the modulator domain.
  • the modulator is inactive when coupled to the cationic domain and the lipophilic domain, and activated when released from the cationic domain.
  • 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.
  • target cells may be defined by a targeting moiety that directs the compound to certain cells.
  • a lipophilic group to further improve transmembrane delivery has not been suggested.
  • the modulator domain is linked to the cationic domain and the lipophilic domain by a cleavable linker.
  • 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-.
  • the lipophilic domain of the three-domain compound is a fatty acid
  • 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
  • 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-docosa
  • the cationic domain is a peptide and lipophilic domain is a fatty acid.
  • the cationic domain is a peptide and lipophilic domain is a fatty acid is conjugated to the peptide at a lysine residue.
  • 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
  • K is lysine
  • R is arginine
  • F is phenylalanine
  • G is glycine
  • Q is glutamine
  • P proline
  • V valine
  • Tat is GRKKRRQRRRPPQ
  • NIs is PKKKRKV
  • Lys(Deca) is N ⁇ -decanoyl lysine
  • Lys(Octa) is N ⁇ -octanoyl lysine
  • Lys(Dodeca) is N ⁇ -dodecanoyl lysine
  • Lys(Hexadeca) is N ⁇ -hexadecanoyl lysine and wherein the nucleotide sequence is PNA.
  • 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.
  • the biological membrane is the tight junctions of the blood brain barrier.
  • the biological membrane is a cellular membrane.
  • the delivery across a biological membrane occurs in vivo. In another preferred embodiment, the delivery across a biological membrane occurs ex vivo.
  • 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:
  • 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.
  • the three-domain compounds of the invention can be used to modulate the activity of cellular components, they are useful as medicaments.
  • 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.
  • Yet another aspect of the invention is a commercial kit of parts comprising at the three-domain compound.
  • 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:
  • Fig. 1 Relative cellular uptake of the PNA conjugates in HeLa pLuc705 cells.
  • 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.
  • CPP cell penetrating peptide
  • Fig. 7 Antisense splice correction effect of a cholesteryl-octaarginine PNA conjugate.
  • Fig. 8 A PNA-Cat ⁇ p conjugate in which the PNA is partially complementary to the siRNA antisense strand is used to form the siRNA-Cat ⁇ p hybridization complex.
  • Fig. 9 Sequence of the anti luciferase antisense siRNA and the PNA-Cat ⁇ p 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.
  • 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 ⁇ -amino group of a lysine. This lysine was incorporated using ⁇ - 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.
  • 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.
  • HPLC reversed -phase high performance liquid chromatography
  • 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
  • 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 ⁇ g/mL) (antibiotics) and 10 % fetal calf serum (Gibco, Invitrogen, Denmark). Cell cultures were maintained at 370 C in a humidified atmosphere containing 5 % CO 2 (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 ⁇ l_ and at cell densities of 50-100 x 10 3 cells/ml unless otherwise stated.
  • DMEM Dulbecco ' s modified Eagle ' s medium
  • penicillin/streptomycin 10 ⁇ g/mL
  • 10 % fetal calf serum Gibco, Invitrogen, Denmark.
  • Cell cultures were maintained at 370 C in a humidified atmosphere
  • 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 ⁇ l 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 ⁇ l_ of a lOO ⁇ M 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.
  • luciferase activity was measured by use of the Bright-GloTM Luciferase Assay System (Promega, Ramcon A/S, Birker ⁇ d, Denmark) and a Victor2 Multilabel Counter (Wallac, Perkin-Elmer Danmark A/S, Aller ⁇ d, Denmark) according to manufacturers instructions.
  • RT-PCR program was as follows: [(55 0 C, 35 min), 1 cycle; (95 0 C, 15 min), 1 cycle; (94 0 C, 0.5 min; 55 0 C, 0.5 min; 72 0 C, 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, Hiller ⁇ d, Denmark) and analyzed by L)N- SCAN-IT software (Silk Scientific Corporation, USA).
  • 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.
  • Tat-Deca- PNA and its mismatch PNA were transfected to the cells at the designated concentrations (0-8 ⁇ M).
  • PNAs ((RK) 3 -P
  • 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.
  • 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.
  • Tat-Deca-PNA conjugate is slightly less active than the Arg-Deca- PNA conjugates at lower concentrations, it exhibits the highest activity at 8 ⁇ M 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 ⁇ M.
  • 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.
  • 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.
  • 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 ⁇ l/ml).
  • the p53R cells were transfected with siRNA (40 nM), LFA2000 (2 ⁇ l/ml) and CatLip peptide (2 ⁇ M). Luciferase activity was measured 48 h after transfection. It is seen that Deca-R10 and Stearyl-R8 has activity not dramatically different from LFA2000.
  • oligonucleotide antisense and RNA interference agents have been used for cellular delivery of oligonucleotide antisense and RNA interference agents through chemical conjugation of the peptide to the oligonucleotide.
  • 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.

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Abstract

La présente invention est basée sur la découverte d'après laquelle l'administration transmembranaire d'un agent biologique couplé à un domaine cationique peut être encore améliorée par le couplage à un domaine lipophile, tel qu'un acide gras. Ainsi, un aspect de la présente invention porte sur des composés à trois domaines permettant une administration transmembranaire améliorée. D'autres aspects sont l'utilisation des composés à trois domaines pour une thérapie, des compositions pharmaceutiques comprenant le composé à trois domaines, ainsi que des procédés pour améliorer l'administration transmembranaire.
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WO2009149214A3 (fr) * 2008-06-03 2010-03-11 Aileron Therapeutics, Inc. Compositions et procédés pour améliorer le transport cellulaire de biomolécules
WO2010023346A1 (fr) 2008-08-29 2010-03-04 Universidade De Satiago De Compostela Composés hybrides tripyrrol-octaarginine et utilisation de ceux-ci en tant que médicament pour traiter le cancer et les maladies microbiennes
US20110237499A1 (en) * 2008-08-29 2011-09-29 Eugenio Vazquez Sentis Hybrid tripyrrole-octaarginine compounds and their use as medicament in the treatment of cancer and microbial illnesses
EP2336108A4 (fr) * 2008-08-29 2012-02-15 Univ Santiago Compostela Composés hybrides tripyrrol-octaarginine et utilisation de ceux-ci en tant que médicament pour traiter le cancer et les maladies microbiennes
WO2010039088A1 (fr) * 2008-09-16 2010-04-08 Kariem Ahmed Peptides de pénétration cellulaire chimiquement modifiés pour une administration améliorée de composés de modulation de gène
US20110212028A1 (en) * 2008-09-16 2011-09-01 Kariem Ahmed Chemically modified cell-penetrating peptides for improved delivery of gene modulating compounds
JP2012502985A (ja) * 2008-09-16 2012-02-02 カリエム・アーメド 遺伝子調節化合物の改良された送達のための化学的に修飾された細胞透過性ペプチド
JP2015017125A (ja) * 2008-09-16 2015-01-29 カリエム・アーメド 遺伝子調節化合物の改良された送達のための化学的に修飾された細胞透過性ペプチド
US20140140929A1 (en) * 2008-09-16 2014-05-22 Kariem Ahmed Chemically modified cell-penetrating peptides for improved delivery of gene modulating compounds
CN102811744A (zh) * 2008-09-16 2012-12-05 卡里姆·阿梅德 用于改善基因调节化合物的递送的化学修饰的细胞渗透性肽
EP2350296A4 (fr) * 2008-11-17 2013-04-03 Enzon Pharmaceuticals Inc Lipides cationiques ramifiés pour système d'administration d'acides nucléiques
JP2012509258A (ja) * 2008-11-17 2012-04-19 エンゾン ファーマシューティカルズ，インコーポレーテッド 核酸送達系のための分岐カチオン性脂質
JP2012509272A (ja) * 2008-11-17 2012-04-19 エンゾン ファーマシューティカルズ，インコーポレーテッド 核酸送達系のための放出性カチオン脂質
EP3252068A2 (fr) 2009-10-12 2017-12-06 Larry J. Smith Procédés et compositions permettant de moduler l'expression génique à l'aide de médicaments à base d'oligonucléotides administrés in vivo ou in vitro
EP4089169A1 (fr) 2009-10-12 2022-11-16 Larry J. Smith Procédés et compositions permettant de moduler l'expression génique à l'aide de médicaments à base d'oligonucléotides administrés in vivo ou in vitro
EP3087182A4 (fr) * 2013-12-23 2017-08-23 Memorial Sloan Kettering Cancer Center Méthodes et compositions pour le traitement du cancer utilisant des agents à base d'acides nucléiques de peptides
US10113169B2 (en) * 2013-12-23 2018-10-30 Memorial Sloan-Kettering Cancer Center Methods and compositions for treating cancer using peptide nucleic acid-based agents
EP3581653A3 (fr) * 2013-12-23 2020-02-19 Memorial Sloan-Kettering Cancer Center Procédés et compositions pour le traitement du cancer au moyen d'agents à base d'acides nucléiques de peptides
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US11518996B2 (en) 2013-12-23 2022-12-06 Memorial Sloan-Kettering Cancer Center Methods and compositions for treating cancer using peptide nucleic acid-based agents
WO2018013924A1 (fr) * 2016-07-15 2018-01-18 Massachusetts Institute Of Technology Nanoparticules synthétiques pour l'administration de composés immunomodulateurs
US10300145B2 (en) 2016-07-15 2019-05-28 Massachusetts Institute Of Technology Synthetic nanoparticles for delivery of immunomodulatory compounds
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