WO2003059394A1 - Composition for transporting oligonucleotides through the blood brain barrier and their use for treating central nervous system diseases - Google Patents

Abstract

The invention concerns a compound consisting of at least an oligonucleotide bound to at least a vector peptide capable of transporting it through the blood brain barrier (BBB). The invention also concerns the preparation of said compounds and pharmaceutical compositions containing same useful for preventing and/or treating diseases of the central nervous system.

Description

         <Desc/Clms Page number 1> COMPOSITIONS FOR THE VECTORIZATION OF OLIGONUCLEOTIDES THROUGH THE HEMATOENPHALIC BARRIER AND THEIR USE FOR THE TREATMENT OF DISEASES OF THE CENTRAL NERVOUS SYSTEM. The present invention relates to a compound consisting of at least one oligonucleotide linked to at least one vector peptide capable of transporting the oligonucleotides across the blood-brain barrier (BBB). The invention also relates to the preparation of these compounds and the pharmaceutical compositions containing them useful for the treatment of diseases of the central nervous system. Antisense oligonucleotides constitute a new class of therapeutic agents which have aroused interest in the fight against numerous diseases. Indeed, they have the ability to inhibit the expression of a gene associated with the disease, according to a mechanism specifically involving certain genetic sequences [Crooke, Exp. Opinion. Invest. Drugs (1996) 14:376-387; Temsamani, J. & Guinot, P., Biotechnol. Appl. Biochem (1997) 26, 65-71]. Gene expression is inhibited by hybridization of the oligonucleotide to target messenger RNA (mRNA) sequences by base pairing, a mechanism described by Watson and Crick. These simple base-pairing rules govern the interaction between the antisense oligonucleotide and the target, allowing the design of oligonucleotides capable of targeting any gene whose sequence is known. A major advantage of this strategy over conventional drugs is the potential specificity of the action of antisense oligonucleotides. In principle, an oligonucleotide with a specific sequence of 17 nucleotides or more can be designed to specifically recognize any gene isolated from the human genome. Moreover, the researchers believe that an inhibition at the genetic level is potentially much more effective than at the protein level in fighting a disease. However, the phosphodiester oligonucleotides which exist in the natural state are liable to be degraded by nucleases present in biological media and inside cells, which limits their use as therapeutic agents. Several modified structures of phosphodiester oligonucleotides have therefore been synthesized and tested both in vitro and in vivo. The goal of this work was to increase the half-life of the oligonucleotide while maintaining its ability to bind with a high degree of affinity and selectivity to its RNA target. Although in principle each of the three components of an oligonucleotide, the base, the sugar and the phosphodiester bond, can be modified, the most important modifications have been made on the bond. Phosphorothioate oligonucleotides, in which one of the oxygen atoms of the phosphodiester bond is replaced by a sulfur atom, are of very particular interest [Agrawal, TIBTECH (1996) 14; 376-387]. This relatively simple modification dramatically improved the resistance of the antisense to nucleases. In recent years, phosphorothioate oligonucleotides have been the most widely used analogs and many of them are the subject of clinical trials [Agrawal, TIBTECH (1996) 14; 376-387; Temsamani, J. & Guinot, P., Biotechnol. Appl. Biochem (1997) 26.65-71]. The knowledge acquired during these tests led the researchers to design more chemically elaborated oligonucleotides. Another parameter that can influence the activity of the oligonucleotide is its internalization and its passage through cell membranes. Many internalization studies have shown that these oligonucleotides cross cell membranes very little (Lebleu, B. <Desc/Clms Page number 3> et al., Ciba Found Symp 1997; 209: 47-54; Stein, CA. & Narayanan., Curr Opin Oncol 1994 6:587-94) and generally do not cross the blood-brain barrier. Indeed, several pharmacokinetic and biodistribution studies have shown that oligonucleotides accumulate in most organs except the brain (Beng et al., Antisense Nucleic Acid Drug Dev 2001.11: 15-27; Agrawal et al., Proc Natl Acad Sci U S A 1991;88:7595-9). Consequently, several strategies have been proposed to improve the properties of oligonucleotides: - Synthesis of modified oligonucleotides to improve the physicochemical properties of the molecule (Hughes, et al., In Delivery Strategies for Antisense Oligonucleotides Therapeutics, Edited by S. Akhtar; 1995, CRC Press). Most of these modifications have been found to be toxic and/or decrease the affinity of the oligonucleotide to its RNA target. - Use of particles such as liposomes or nanoparticles (Juliano & Akhtar (1992) Antisense Res Dev 2: 165; Chavany et al 1992; Pharm Res 9: 441). Although interesting results have been obtained in vitro, in vivo results have been disappointing. - Conjugation of the oligonucleotide to monoclonal antibodies such as OX26 (Boado, et al., J Pharm Sci 1998 87: 1308-1315) or to polymers such as polylysine (Leonetti, et al., 1990; Bioconj. Chem 1:149). The coupling of these macromolecules is complex and can lead in certain cases to the development of immunogenicity in animals. The Applicant has demonstrated that linear peptide vectors, such as linear peptides derived from natural peptides such as Protegrin and Tachyplesin transport active molecules through <Desc/Clms Page number 4> the BBB and improve the pharmacological properties of these molecules. The work and results concerning these linear peptides and their use as vectors of active molecules have been described in French patent applications N 98/15074 filed on November 30, 1998 and in French patent application N 99/02938 filed on November 26, 1999 The Applicant has now demonstrated that these linear peptides are capable of very significantly increasing the penetration of oligonucleotides through the blood-brain barrier. A subject of the present invention is therefore compounds consisting of at least one oligonucleotide linked to at least one linear peptide capable of vectorizing said oligonucleotide across the blood-brain barrier. More particularly, these linear peptides are derivatives of protegrins or tachyplesins. Protegrins and Tachyplesins are natural antibiotic peptides whose structure is of the hairpin type maintained by disulphide bridges. These bridges play an important role in the cytolytic activity observed on human cells. The term protegrins refers to a set of five peptides designated PG-1, PG-2, PG-3, PG-4 and PG-5 whose sequences are given below, closely related and isolated from porcine leukocytes ( V. N. Kokryakov & col. 1993, FEBS lett. 327, 231-236): PG-1: RGGRLCYCRRRFCVCVGR-NH2 PG-2: RGGRLCYCRRRFCICV-NH2 PG-3: RGGGLCYCRRRFCVCVGR-NH2 PG-4: RGGRLCYCRGWICFCVGR-NH2 PG-5: RGGRLCYCRPRFC VCVGR -NH2 <Desc/Clms Page number 5> or said peptides of formula (I) or (II), in retro form, consisting of amino acids of D and/or L configuration, or a fragment thereof consisting of a sequence of at least 5 and preferably at least 7 successive amino acids of the peptides of formulas (I) or (II). In a second form of implementation, the invention envisages most particularly as linear peptides derived from Protegrins, a peptide which corresponds to the following formula (I): BX (X or B) BXXXXBBBXXXXXXB (I) and by linear peptide derived from Tachyplesins, a peptide which corresponds to the following formula (II): BXXXBXXXBXXXXBBXB (II), in which: - B is chosen from arginine, lysine, diaminoacetic acid, diaminobutyric acid, diaminopropionic acid, l ornithine. - X is chosen from glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteine, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenylalanine, histidine, tryptophan, tyrosine, proline, Abu, amino-1-cyclohexane carboxylic acid, Aib, 2-aminotetralin carboxylic, 4-bromophenyalanine, tert-Leucine, 4 -chlorophenylalanine, beta-cyclohexylalanine, 3,4-dichlorophenyalanine, 4-fluorophenyalanine, homoleucine, beta-homoleucine, homophenyalanine, 4-methylphenyalanine, 1-naphthyalanine, 2-naphthyalanine, 4 -nitrophenyalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridyalanine, [2-thienyl]alanine, <Desc/Clms Page number 6> Tachyplesins (Tamura, H. et al., 1993, Chem. Pharm. Bul. Tokyo 41,978-980), designated T1, T2 and T3 and the polyphemusins (Muta, T., 1994, CIBA Found. Sym. 186, 160-174), designated P1 and P2, the sequences of which are given below below are homologous peptides isolated from the hemolymph of two crabs, Tachyplesus tridentatus for tachyplesins T1, T2 and T3 and Limmulus polyphemus for polyphemusins P1 and P2: PI: RRWCFRVCYRGFCYRKCR-NH2 P2: RRWCFRVCYKGFCYRKCR-NH2 Protegrins, tachyplesins and polyphemusins contain a high proportion of basic residues (lysines and arginines) and have four cysteines which form two parallel disulphide bridges. These three families of peptides also show homologies with certain defensins and in particular with the human defensin NP-1 (Kokryakov, V.

   N. et al., 1993, Febs Let. 327,231-236). In a first embodiment, the invention particularly envisages as linear peptides derived from Protegrins, a peptide which corresponds to the following formula (I): BX (X or B) BXXXXBBBXXXXXXB (I) and by linear peptide derived from Tachyplesins, a peptide which corresponds to the following formula (II): BXXXBXXXBXXXXBBXB (II), in which: - the B groups, which are identical or different, represent an amino acid residue whose side chain bears a basic group, and - the groups X, identical or different, represent an aliphatic or aromatic amino acid residue, <Desc/Clms Page number 7> or the said peptides of formulas (I) or (II), in retro form, consisting of amino acids of configuration D and/or L, or a fragment thereof consisting of a sequence of at least 5 and preferably of at least 7 successive amino acids of the peptides of formulas (I) or (II). As examples of oligonucleotides used in the context of the compounds of the invention, mention may be made of oligonucleotides phosphodiesters, phosphorothioates, methylphosphonates, hybrids comprising an RNA part and a DNA part, PNAs (Peptide Nucleic Acids), etc They are advantageously antisense oligonucleotides as defined above. The bond between the oligonucleotide and the linear peptide in the compounds of the invention is chosen from a covalent bond, a hydrophobic bond, an ionic bond, a cleavable bond or a non-cleavable bond in physiological media or inside cells. This link can be direct or indirect by means of a linker and carried out by means of a functional group naturally present or introduced either on the peptide, or on the oligonucleotide, or on both. This linker, if present, must be acceptable taking into account the chemical nature and the size of both the peptide and the oligonucleotide. Mention may be made, by way of non-exhaustive example of such linkers, of molecules containing bonds of the amide, ester, carbamate, carbonate, disulphide, anhydride, hydrazone, hydrazine, imine, ether, amine type, an alkyl, aryl, alkylaryl as well as their various unsaturated or polyunsaturated derivatives. This linker can be branched and carry <Desc/Clms Page number 8> various functionalized or non-functionalized substituents such as alcohol, amine, carboxyl, sulfhydryl. As functional groups, mention may be made of: -OH, -SH, -COOH, or -NH2. Thus, the oligonucleotide(s) can be linked by covalent bonds at the N-terminal or C-terminal ends or else at the side chains of the peptide. The linear peptide(s) can be linked to the oligonucleotide, at its 3′-OH and/or 5′-OH function or else at another site such as the base or the sugar. A preferred type of bond between the oligonucleotide(s) and the linear peptide(s) involves at least one disulfide bridge. Indeed, this type of bond is characterized by its stability in the plasma after injection of the compound, then once the compounds of the invention have crossed the blood-brain barrier, said disulphide bridge is reduced by releasing the oligonucleotide. The link can be made at any site of the peptide as indicated above. The present invention also relates to a method for treating diseases of the central nervous system (CNS), such as brain cancer or neurodegenerative diseases, consisting in administering to a subject suffering from such a disease an effective amount of a compound described previously. The invention therefore relates to a pharmaceutical composition for the treatment of diseases of the central nervous system comprising, as active agent, at least one compound described above. For the treatment of these diseases, mention may be made of the following target genes: protein kinase for cancer, amyloid beta protein for Alzheimer's disease, TNF-alpha for neuroprotection, the dopamine receptor for Parkinson's disease. <Desc/Clms Page number 9> A particular embodiment of the invention consists in combining, in the composition, the compound described previously with one or more substances which are useful for the treatment of diseases of the CNS. Examples of such substances include an anticancer agent, such as carmustine for the treatment of brain cancer, L-Dopa for the treatment of Parkinson's disease, or galantamine for the treatment of Parkinson's disease. Alzheimer's. A pharmaceutical composition according to the invention is in a form suitable for administration by the intravenous route, by the subcutaneous route, by the intramuscular route, orally, by the rectal route, by the nasal route, by the transdermal route, by the pulmonary route. Other advantages and characteristics of the invention will appear on reading the examples which follow concerning the preparation of a compound consisting of an oligonucleotide and a linear peptide, its penetration and its activity. Reference will be made to the appended drawings in which: FIG. 1 schematically represents the chemical synthesis of a vectorized compound of the oligonucleotide. - Figure 2 illustrates the cell penetration results comparing the internalization between compound 1 (oligonucleotide alone) and compound 2 (vectorized oligonucleotide). - Figure 3 illustrates the passage through the blood-brain barrier of compound 1 (oligonucleotide), compound 2 (vectorized oligonucleotide). I-Chemical synthesis of the vectorized oligonucleotide. <Desc/Clms Page number 10> 1) Synthesis of the vector peptide. The SynB3 peptide of formula SEQ ID NO. 1: RRLSYSRRRF is assembled on a solid phase according to an Fmoc/tu strategy, cleaved and deprotected with trifluoroacetic acid, then purified by preparative high pressure reverse phase chromatography and lyophilized. Its purity (> 95%) and its identity are confirmed by analytical HPLC and by mass spectrometry. 2) Synthesis of the oligonucleotide. The phosphodiester oligonucleotide complementary to the c-myc RNA was assembled on solid phase (Biosearch 8700, Milligen). After deprotection, the oligonucleotide was purified by ion-exchange HPLC and desalted by a SepPak C18 column. In the case of cell penetration experiments, a fluorescent group (Fluorescine) was added to the oligonucleotide. 3) Coupling of the oligonucleotide on SynB3. The phosphodiester oligonucleotide (1. equivalent) is solubilized in PBS buffer (500 μl) and the SynB3 vector (2 equivalents) is solubilized in DMF (100 μl). The vector peptide is added slowly to the oligonucleotide solution and the mixture is incubated for approximately 8 hours at 55° C. The yield of the reaction is measured by analysis of an aliquot by mass spectroscopy. The product is then purified by semi-preparative HPLC (10mm/250mm C18 column) by monitoring the absorbance at 215 nm, 280 nm and 480 nm and 525 nm (for the fluorescence). The gradient used is from 0 to 60% acetonitrile in 60 min. II-Compounds tested. The compounds tested are given in Table 1 below. <Desc/Clms Page number 11> Table 1 EMI11.1 <tb> Compound <SEP> 1 <SEP> 5'-GTG <SEP> CCG <SEP> GGG <SEP> TCT <SEP> TCG--GGC-3' <tb> <tb> Compound <SEP> 2 <SEP> RRLSYSRRRF-S-S-GTGCCGGGGTCTTCGGGC <tb> III-Assays used. 1) Cellular Penetration. The cellular penetration of the compounds was studied by flow cytometry. The K562 cells (obtained from the ATCC) are cultured in RPMI medium with 10% fetal calf serum. The cells are diluted to 0.3 x 106 cells per ml, 24 hours before the experiment. Cell penetration was measured by flow cytometry using a FACScan (Becton Dickinson, USA). The fluorescent compounds (final concentration 5 μM) are incubated with the K562 cells (5×105 cells per ml) in an Optimem medium at 37° C. for variable times (the final volume was 0.5 ml). After the incubation, the cells are washed twice and then re-suspended in 0.5 ml of cold PBS. Penetration was then analyzed by FACS. The fluorophores are excited at 488 nm and the fluorescence is measured at 525 nm. A histogram of fluorescence intensity (for 1X10 4 cells) is obtained and the mean distribution was taken as representative of the amount of cell-associated peptide. Results are shown as the percentage of cells that incorporated fluorescence. 2) Marking at the 3'-terminal. The free or vectorized oligonucleotide was labeled at its 3′-OH terminal using the terminal transferase enzyme (Promega) following the protocol described by the supplier <Desc/Clms Page number 12>. Briefly, approximately 10 pmoles of compound are inbubed with the terminal transferase at 37 C in the presence of dideoxyadenosine triphosphate radiolabeled with phosphorus 32 (32P-ddATP). After 30 min of incubation, the reaction is stopped by heating to 65° C. The radiolabeled product is then separated from the free labeled nucleotide using a gel exclusion column (spin column, Qiagen). 3) In situ Brain Perfusion Test. Mice (20-25 g, Iffa-Credo; l'Arbresle, France) are anesthetized. After exposure of the common carotid, the right external carotid artery is linked at the level of the bifurcation with the internal carotid and the common carotid is linked between the heart and the site of implantation of the catheter (polyethylene catheter, ID: 0.76). This, previously filled with a heparin solution (100 units/ml), is inserted into the common carotid artery. The mice are perfused with the perfusion buffer (128 mM NaCl, 24 mM NaHCO3, 4.2 mM KCl, 2.4 mM NaH2PO4, 1. 5 mM CaCl2, 0.9 mM MgSO4, and 9 mM D-glucose). This buffer is filtered and then bubbled with a mixture containing 95% 2/5% C 2 in order to maintain the pH close to 7.4 and to supply the brain with oxygen during the perfusion. The mice are perfused with the buffer containing the free oligonucleotide or the vectorized oligonucleotide. In each product the oligonucleotide is radiolabeled with 32p. Just before the start of the perfusion, the heart is stopped by section of the ventricles, in order to avoid reflux of the perfusate during the perfusion. The right hemisphere is then perfused at a rate of 10 ml/min for 60 seconds after which the mouse is decapitated. The amount of radioactivity in the right hemisphere is then measured and the cerebral penetration (Kin) is calculated. <Desc/Clms Page number 13> IV-Results. 1) Cell Penetration We first compared the internalization of the free oligo (compound 1) with that of the vectorized oligonucleotide (compound 2). Both products were labeled with fluorescine at the 3'-OH of the oligonucleotide. The results represented in FIG. 2 show the internalization kinetics of the two products. The free oligonucleotide has very low penetration. Thus, after 90 min of incubation, only 3% of the cells are fluorescent. On the other hand, for the vectorized oligonucleotide, approximately 60% of the cells incorporated the oligonucleotide. This indicates that the vectorization makes it possible to significantly improve the internalization of the oligonucleotide. 2) Penetration into the brain. In this study, we compared the penetration into the BBB of the free oligonucleotide with that of the vectorized oligonucleotide. Both products were infused into the mouse brain. After 60 seconds of perfusion in the buffer, the penetration of the products is estimated by the influx constant or Kin in μl/sec/g. brain approximately 3 times after a 60 second infusion in buffer.
    

Claims

CLAIMS 1) A compound consisting of at least one oligonucleotide linked to at least one linear peptide derived from protegrins or tachyplesins.

2) A compound according to claim 1, characterized in that the linear peptide is chosen from those of formulas (I) or (II) below: BX (X or B) BXXXXBBBXXXXXXB (I) BXXXBXXXBXXXXBBXB (II), in which: - the B groups, which are identical or different, represent an amino acid residue whose side chain bears a basic group, and - the X groups, which are identical or different, represent an aliphatic or aromatic amino acid residue or said peptides of formula (I) or (II), in retro form, consisting of amino acids of D and/or L configuration, or a fragment thereof consisting of a sequence of at least 5 and preferably of at least 7 successive amino acids of the peptides of formulas (I) or (II).

3) A compound according to claim 1, characterized in that the linear peptide is chosen from those of formulas (I) or (II) below: BX (X or B) BXXXXBBBXXXXXXB (I) BXXXBXXXBXXXXBBXB (II), in which: B is selected from arginine, lysine, diaminoacetic acid, diaminobutyric acid, diaminopropionic acid, ornithine, <Desc/Clms Page number 15> X is selected from glycine, alanine, valine, norleucine, isoleucine, leucine, cysteine, cysteine, penicillamine, methionine, serine, threonine, asparagine, glutamine, phenyalanine, histidine, tryptophan, tyrosine, proline, Abu, amino-1-cyclohexane carboxylic acid, Aib, 2-aminotetralin carboxylic, 4-bromophenyalanine,

tert-Leucine, 4-chlorophenylalanine, beta-cyclohexylalanine, 3,4-dichlorophenyalanine, 4-fluorophenyalanine, homoleucine, beta-homoleucine, homophenyalanine, 4-methylphenyalanine, 1-naphthyalanine, 2-naphthyalanine, 4-nitrophenyalanine, 3-nitrotyrosine, norvaline, phenylglycine, 3-pyridyalanine, [2-thienyl] alanine, or said peptides of formulas (I) or (II), in retro form, consisting of amino acids of configuration D and / or L, or a fragment thereof consisting of a sequence of at least 5 and preferably at least 7 successive amino acids of the peptides of formulas (I) or ( II).

4) A compound according to any one of claims 1 to 3, characterized in that the oligonucleotide is chosen from oligonucleotides phosphodiesters, phosphorothioates, methylphosphonates, hybrids comprising an RNA part and a DNA part, the PNAs.

5) A compound according to any one of the preceding claims, characterized in that the oligonucleotide is an antisense.

6) A compound according to any one of the preceding claims, characterized in that the bond between the oligonucleotide and the linear peptide is <Desc/Clms Page number 16> chosen from a covalent bond, a hydrophobic bond, an ionic bond, a cleavable bond or a non-cleavable bond in physiological media or inside cells.

7) A compound according to any one of the preceding claims, characterized in that the bond between the oligonucleotide and the linear peptide is a direct or indirect bond via a linker arm.

8) A compound according to any one of the preceding claims, characterized in that the bond between the oligonucleotide and the linear peptide is carried out by means of a functional group naturally present or introduced either on the peptide, or on the oligonucleotide , or both.

9) A compound according to any one of the preceding claims, characterized in that the oligonucleotide(s) are linked by covalent bonds at the N-terminal or C-terminal ends or else at the level of the side chains of the peptide(s).

10) A compound according to any one of the preceding claims, characterized in that the linear peptide or peptides are linked to the oligonucleotide, at its 3'-OH and/or 5'OH functions or else at another site such as the base or the sugar.

11) A pharmaceutical composition for the treatment of diseases of the central nervous system, characterized in that it comprises as active agent at least one compound according to any one of claims 1 to 10. <Desc/Clms Page number 17>

12) A pharmaceutical composition for the treatment of diseases of the central nervous system according to claim 11, characterized in that it further comprises one or more other substances useful for the treatment of diseases of the central nervous system.

13) Use of a linear peptide as defined in any one of claims 1 to 10 for the preparation of a medicament intended for the treatment and / or prevention of diseases of the central nervous system, said peptide being linked to at least an oligonucleotide to vectorize it through the BBB.