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Title:
ENCAPSULATION OF PHENAZINE AND DERIVATIVES THEREOF
Document Type and Number:
WIPO Patent Application WO/2019/175163
Kind Code:
A1
Abstract:
The present invention relates to a nanoparticle comprising a modified oligonucleotide in the form of a micelle, loaded with an active compound selected from phenazine and derivatives thereof, wherein the modified oligonucleotide is an oligonucleotide modified by substitution at the 3' or the 5' end by a moiety comprising: - either at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, linear or branched, hydrocarbon chains comprising from 1 to 22 carbon atoms, - or at least three saturated or unsaturated, linear or branched hydrocarbon chains comprising from 2 to 30 carbon atoms.

Inventors:
BARTHELEMY, Philippe (UNIVERSITE DE BORDEAUX - INSERM U1212 146 RUE LEO SAIGNAT CAMPUS CARREIRE BAT3, BORDEAUX, 33000, FR)
BENIZRI, Sébastien (ChemBioPharm / Inserm U1212 - CNRS UMR 5320 / 146 Rue Léo-Saignat, Carreire Bat. Pharmacie - 3e tranche 4e étage, BORDEAUX, 33076, FR)
BRANGER, Nicolas (Laboratoire oncologie predictive 27 Boulevard Lei Roure BP, 13273 MARSEILLE CEDEX 09, 13273, FR)
CAMPLO, Michel (Aix Marseille Université - CINaM UMR 7325 - CNRS - Campus de Luminy Case 913, MARSEILLE CEDEX 09, 13288, FR)
KARAKI, Sara (INSTITUT PAOLI CALMETTES - U1068 27bd Lei Roure CS, 13273 MARSEILLE CEDEX 09, 13273, FR)
ROCCHI, Palma (INSTITUT PAOLI CALMETTES - U1068 27bd Lei Roure CS, 13273 MARSEILLE CEDEX 09, 13273, FR)
SIRI, Olivier (Aix Marseille Université - CINaM UMR 7325 - CNRS - Campus de Luminy Case 913, MARSEILLE CEDEX 09, 13288, FR)
Application Number:
EP2019/056146
Publication Date:
September 19, 2019
Filing Date:
March 12, 2019
Export Citation:
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Assignee:
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM) (101 Rue de Tolbiac, PARIS, PARIS, 75013, FR)
INSTITUT JEAN PAOLI & IRENE CALMETTES (Centre Régional de Lutte Contre le Cancer 232 boulevard de Sainte-Marguerite, MARSEILLE, 13009, FR)
UNIVERSITÉ D'AIX-MARSEILLE (Jardin du Pharo 58, boulevard Charles Livon, MARSEILLE, 13007, FR)
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (3 rue Michel Ange, PARIS, PARIS, 75016, FR)
International Classes:
C07H21/00; A61K31/7088; A61P35/00; B82Y5/00; C07D241/46
Domestic Patent References:
WO2011117830A12011-09-29
WO2014195430A12014-12-11
WO2014195432A12014-12-11
WO2011117830A12011-09-29
WO2014195430A12014-12-11
WO2014195432A12014-12-11
Other References:
OLEKSANDR POKHOLENKO ET AL: "Lipid oligonucleotide conjugates as responsive nanomaterials for drug delivery", JOURNAL OF MATERIALS CHEMISTRY B, vol. 1, no. 39, 1 January 2013 (2013-01-01), pages 5329, XP055101830, ISSN: 2050-750X, DOI: 10.1039/c3tb20357c
FIRE ET AL., NATURE, vol. 391, 1998, pages 806 - 811
CARTHEW ET AL., CURRENT OPINIONS IN CELL BIOLOGY, vol. 13, 2001, pages 244 - 248
ELBASHIR ET AL., NATURE, vol. 411, 2001, pages 494 - 498
ZENG ET AL., METHODS ENZYMOL., vol. 392, 2005, pages 371 - 380
BERGE ET AL., J. PHARM. SD, vol. 66, 1977, pages 1
TETRAHEDRON LETTERS, vol. 56, no. 21, 20 May 2015 (2015-05-20), pages 2695 - 2698, Retrieved from the Internet
Attorney, Agent or Firm:
DOMENEGO, Bertrand et al. (LAVOIX, 2 place d'Estienne d'Orves, PARIS CEDEX 09, 75441, FR)
Download PDF:
Claims:
CLAIMS

1. A nanoparticle comprising a modified oligonucleotide in the form of a micelle, loaded with an active compound selected from phenazine and derivatives thereof,

wherein the modified oligonucleotide is an oligonucleotide modified by substitution at the 3’ or the 5’ end by a moiety comprising:

- either at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, linear or branched, hydrocarbon chains comprising from 1 to 22 carbon atoms,

- or at least three saturated or unsaturated, linear or branched hydrocarbon chains comprising from 2 to 30 carbon atoms.

2. The nanoparticle of claim 1 , wherein the modified oligonucleotide has the general formula (I) or (II):

Oligo

Oligo

(I)

wherein:

• Oligo represents an oligonucleotide sequence which may be oriented 3’-5’ or 5’-3’, simple and/or double stranded, DNA, RNA, and/or comprise modified nucleotides;

• X represents a divalent linker moiety selected from -0-, -S-, -NH-, and -CH2-;

• Ri and R2 may be identical or different and represent:

(i) a hydrogen atom,

(ii) a halogen atom, in particular fluorine atom,

(iii) a hydroxyl group,

(iv) an alkyl group comprising from 1 to 12 carbon atoms; • Li and l_2 may be identical or different and represent a saturated or unsaturated, linear or branched hydrocarbon chain comprising from 1 to 22 carbon atoms,

• B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms;

• Mi, M2 and M3 may be identical or different and represent:

- a saturated or unsaturated, linear or branched hydrocarbon chain comprising from 2 to 30 carbon atoms, which may be substituted by one or more halogen atoms, notably be fluorinated or perfluorinated and/or be interrupted by one or more groups selected from -0-, -S-, -NH-, -O-C(O)-, -0-C(S)-NH-, -0-C(0)-0-, -0-C(0)-NH-, -0-P(0)(0)-0- and -P-0(0)(0)- groups; and/or be substituted at the terminal carbon atom by an aliphatic or aromatic, notably benzylic or naphtylic ester or ether group;

- an acyl radical with 2 to 30 carbon atoms, or

- an acylglycerol, sphingosine or ceramide group.

3. The nanoparticle according to claim 2, wherein the oligonucleotide is selected from the group consisting of dTi5, dAi5, and an oligonucleotide consisting of the sequence SEQ ID NO: 6.

4. The nanoparticle according to claim 2 or 3, wherein, in formula (I) or (II), X is -0-.

5. The nanoparticle according to any of claims 2 to 4, wherein, in formula (I) or (II), Ri and R2 are hydrogen atoms.

6. The nanoparticle according to any of claims 2 to 5, wherein, in formula (I), Li and U represent a hydrocarbon chain comprising from 6 to 22 carbon atoms, preferably from 8 to 18 carbon atoms, advantageously from 12 to 16 carbon atoms.

7. The nanoparticle according to any of claims 2 to 6, wherein, in formula (I), B is a non-substituted nucleobase selected from the group consisting of: uracil, thymine, adenine, cytosine, 6-methoxypurine, and hypoxanthine.

8. The nanoparticle according to any of claims 2 to 5, wherein, in formula (II), Mi , M2 and M3 represent a hydrocarbon chain comprising from 6 to 22 carbon atoms, preferably from 12 to 20 carbon atoms.

9. The nanoparticle according to any of claims 2 to 8, wherein the oligonucleotide comprises a fragment of at least 10 consecutive nucleotides of a sequence selected from the group consisting of: SEQ ID NO: 2 (5’- ACCAATGAGCGAGTCATCAA-3’), SEQ ID NO: 3 (5’-AACCCGUCCGCGAUCU CCCGG-3’), SEQ ID NO: 6 (5’-AACTTGTTTCCTGCAGGTGA-3’), SEQ ID NO: 7 (5’-T GGTT CAT G ACAAT AT CG AC-3’) , SEQ ID NO: 8 (5’-TAATCATGATGGCGACT GAA-3’), SEQ ID NO: 16 (5’- ACCAGT GATT ACT GTGCTTT -3’) , SEQ ID NO: 17 (5’- CTTGTAGGCTTCTTTTGTGA-3’), SEQ ID NO: 18 (5’-AT GT AAT CTTT GAT GT AC TT-3’), SEQ ID NO: 19 (5’-GTTT CCCTTT GATT G ATTT C-3’) , SEQ ID NO: 20 (5’-TT CTGGTCTCTGTT CTT C AA-3’) , SEQ ID NO: 25 (5’-AGAAAATCATATATGGGGTC- 3’), SEQ ID NO: 27 (5’-TT AACATTT CT CCATTT CT A -3’), SEQ ID NO: 29 (5’-GT CAT AAAAGGTTTT ACT CT-3’) and SEQ ID NO: 31 (5’-GAAATTAGCAAGGATGTG CT-3’).

10. The nanoparticle according to claim 9, wherein the oligonucleotide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 7.

11. The nanoparticle according to any of claims 1 to 10, wherein the active compound is a phenazine derivative having the following formula (III):

wherein Ri , R2, R3 and R4, independently from each other, are selected from the group consisting of:

- a hydrogen atom;

- a halogen atom chosen among chlorine, fluorine, bromine and iodine atom;

- a cyano group;

- -N02;

- a -N(R5)(Re) group, with R5 and R6 representing, independently the one of the other, a hydrogen atom, a linear or branched (Ci-C20)alkyl group, a benzyl group or a -COR10 group; - an amido group;

- a -NHS02(R7) group, R? representing a linear or branched (Ci-C2o)alkyl group or an aryl group;

- a -ORs group, R8 representing H or a linear or branched (Ci-C20)alkyl group;

- a -OCORg group, Rg representing H or a linear or branched (Ci-C20)alkyl group;

- a (CrC2o)alkyl or (Ci-C20)alkylthio group, linear or branched, satured or non satured,

- a -COR10 group, R10 representing a hydroxyl group or a linear or branched (CrC2o)alkoxy group, and

- a -CO-[NH-(CH2)n]m-N(R5)(R6), -HN 0-[(CH2)n-N(Rii)]p-(CH2)n-N(Rii)(R6), or -0-C0-[(CH2)n-N(Rii)]p-(CH2)n-N(Rii)(R6) group,

. R5 and R6 being are such as defined above,

. R1 1 representing a hydrogen atom or a tert-butyloxycarbonyl group,

. p representing an integer ranging from 0 to 1 , and

. n, n' and m, independently, representing an integer with n and n', independently, ranging from 2 to 6 and m ranging from 1 to 2, and, when m is 2, each n value may be identical or different one from another, or anyone of its pharmaceutically acceptable salts.

12. The nanoparticle according to any of claims 1 to 1 1 , wherein the active compound has the following formula:

13. The nanoparticle of any of claims 1 to 12, for use as a medicament.

14. The nanoparticle of any of claims 1 to 13, for use for the treatment of cancer.

15. The nanoparticle for the use of claim 14, wherein the cancer is a prostate cancer.

Description:
ENCAPSULATION OF PHENAZINE AND DERIVATIVES THEREOF

The present invention relates to the encapsulation of phenazine and derivatives thereof, and to the corresponding nanoparticles. It also relates to said nanoparticles for their use for treating cancer.

Numerous researches are dedicated to cancers and to their mechanisms of action.

Prostate cancer (PC) is one of the most common causes of death by cancer in the western world. Once diagnosed, androgen ablation is the first line therapy. However, the disease progresses to a castration-resistant state (CRPC), and chemotherapy used prolongs life span of a few months. In order to improve therapy for CRPC, one strategy is to target genes amplified after androgen deprivation. It has been shown that Translationally controlled Tumor protein (TCTP) is overexpressed and p53 expression and function are lost in CRPC. Analysis showed that TCTP’s expression was found to be significantly down regulated after androgen ablation to become uniformly highly expressed in 75% of castration-resistant prostate cancer.

Phenazine and derivatives thereof, such as those disclosed in the international application WO 201 1/1 17830, are known as anti-cancer agents. However, such compounds have several drawbacks, especially concerning their solubility properties. Indeed, the greatest disadvantage of these compounds remains their very low solubility, notably in aqueous medium and biological fluids. Due to this low solubility, the phenazine derivatives cannot be administered as such to patients.

The aim of the present invention is thus to provide an efficient tool for the delivery of TCTP, together with phenazine and derivatives thereof, especially for treating cancer, in particular for treating castration-resistant prostate cancer.

Another aim of the present invention is to provide an improved treatment of cancer, in particular castration-resistant prostate cancer.

Another aim of the present invention is to provide means for encapsulating phenazine and derivatives thereof, while maintaining the efficiency of the active compound especially for the treatment of prostate cancer, in particular castration- resistant prostate cancer. Thus, the present invention concerns a nanoparticle comprising a modified oligonucleotide in the form of a micelle, loaded with an active compound selected from phenazine and derivatives thereof,

wherein the modified oligonucleotide is an oligonucleotide modified by substitution at the 3’ or the 5’ end by a moiety comprising:

- either at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, linear or branched, hydrocarbon chains comprising from 1 to 22 carbon atoms,

- or at least three saturated or unsaturated, linear or branched hydrocarbon chains comprising from 2 to 30 carbon atoms.

The present invention is thus based on the combination of a modified oligonucleotide and an active compound being phenazine or a derivative thereof. The modified oligonucleotide is in the form of a micelle having a core-shell structure and the phenazine or derivative thereof is thus encapsulated in the core of said micelle.

The modified oligonucleotide thus allows the encapsulation of phenazine or of a derivative thereof.

The present invention thus relates to a nanomicelle consisting in a modified oligonucleotide as defined above, loaded with an active compound as defined above.

The nanoparticles according to the invention have a particle size varying preferably from 1 nm to several hundreds of nanometers.

The nanoparticles according to the invention may be in the form of aggregates. According to an embodiment, said aggregates have a diameter comprised between 50 nm and 400 nm, preferably between 100 and 300 nm. According to an embodiment, said aggregates have a zeta potential comprised between -80 mV and -10 mV, preferably between -70 mV and -40 mV.

Modified oligonucleotide

According to the invention, the nanoparticle as defined above is a micelle of a modified oligonucleotide, said modified oligonucleotide being an oligonucleotide modified by substitution at the 3’ or the 5’ end either by a moiety comprising at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, linear or branched, hydrocarbon chains comprising from 1 to 22 carbon atoms, or by a moiety comprising at least three saturated or unsaturated, linear or branched hydrocarbon chains comprising from 2 to 30 carbon atoms.

Oligonucleotide

As used herein, the term "oligonucleotide" refers to a nucleic acid sequence, 3'-5' or 5'-3' oriented, which may be single- or double-stranded. The oligonucleotide used in the context of the invention may in particular be DNA or RNA.

The oligonucleotides used in the context of the invention may be further modified, preferably chemically modified, in order to increase the stability and/or therapeutic efficiency of the oligonucleotides in vivo. In particular, the oligonucleotide used in the context of the invention may comprise modified nucleotides.

Chemical modifications may occur at three different sites: (i) at phosphate groups, (ii) on the sugar moiety, and/or (iii) on the entire backbone structure of the oligonucleotide.

For example, the oligonucleotides may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atom with a sulfur atom) which have increased resistance to nuclease digestion. 2’-methoxyethyl (MOE) modification (such as the modified backbone commercialized by ISIS Pharmaceuticals) is also effective.

Additionally or alternatively, the oligonucleotides of the invention may comprise completely, partially or in combination, modified nucleotides which are derivatives with substitutions at the 2' position of the sugar, in particular with the following chemical modifications: O-methyl group (2'-0-Me) substitution, 2- methoxyethyl group (2'-0-M0E) substitution, fluoro group (2'-fluoro) substitution, chloro group (2'-CI) substitution, bromo group (2'-Br) substitution, cyanide group (2'- CN) substitution, trifluoromethyl group (2'-CF 3 ) substitution, OCF 3 group (2'-OCF 3 ) substitution, OCN group (2'-OCN) substitution, O-alkyl group (2'-0-alkyl) substitution, S-alkyl group (2'-S-alkyl) substitution, N-alkyl group (2'-N-akyl) substitution, O-alkenyl group (2'-0-alkenyl) substitution, S-alkenyl group (2'-S- alkenyl) substitution, N-alkenyl group (2'-N-alkenyl) substitution, SOCH 3 group (2'- SOCH 3 ) substitution, S0 2 CH 3 group (2'-S0 2 CH 3 ) substitution, 0N0 2 group (2'- 0N0 2 ) substitution, N0 2 group (2'-N0 2 ) substitution, N 3 group (2'-N 3 ) substitution and/or NH 2 group (2'-NH 2 ) substitution. Additionally or alternatively, the oligonucleotides of the invention may comprise completely or partially modified nucleotides wherein the ribose moiety is used to produce locked nucleic acid (LNA), in which a covalent bridge is formed between the 2' oxygen and the 4' carbon of the ribose, fixing it in the 3'-endo configuration. These constructs are extremely stable in biological medium, able to activate RNase H and form tight hybrids with complementary RNA and DNA.

Accordingly, in a preferred embodiment, the oligonucleotide used in the context of the invention comprises modified nucleotides selected from the group consisting of LNA, 2’-OMe analogs, 2’-phosphorothioate analogs, 2’-fluoro analogs, 2’-CI analogs, 2’-Br analogs, 2’-CN analogs, 2’-CF 3 analogs, 2’-OCF 3 analogs, 2’- OCN analogs, 2’-0-alkyl analogs, 2’-S-alkyl analogs, 2’-N-alkyl analogs, 2’-0- alkenyl analogs, 2’-S-alkenyl analogs, 2’-N-alkenyl analogs, 2’-SOCH 3 analogs, 2’- S0 2 CH 3 analogs, 2’-0N0 2 analogs, 2’-N0 2 analogs, 2’-N 3 analogs, 2’-NH 2 analogs and combinations thereof. More preferably, the modified nucleotides are selected from the group consisting of LNA, 2’-OMe analogs, 2’-phosphorothioate analogs and 2’-fluoro analogs.

Additionally or alternatively, some nucleobases of the oligonucleotide may be present as desoxyriboses. That modification should only affect the skeleton of the nucleobase, in which the hydroxyl group is absent, but not the side chain of the nucleobase which remains unchanged. Such a modification usually favors recognition of the iRNA by the DICER complex.

The oligonucleotide according to the invention may for example correspond to antisense oligonucleotides or to interfering RNAs (including siRNAs, shRNAs, miRNAs, dsRNAs, and other RNA species that can be cleaved in vivo to form siRNAs), that preferably target mRNAs of interest.

As used herein, an oligonucleotide that “targets” an mRNA refers to an oligonucleotide that is capable of specifically binding to said mRNA. That is to say, the oligonucleotide comprises a sequence that is at least partially complementary, preferably perfectly complementary, to a region of the sequence of said mRNA, said complementarity being sufficient to yield specific binding under intra-cellular conditions.

As immediately apparent to the skilled in the art, by a sequence that is “perfectly complementary to” a second sequence is meant the reverse complement counterpart of the second sequence, either under the form of a DNA molecule or under the form of a RNA molecule. A sequence is“partially complementary to” a second sequence if there are one or more mismatches.

Preferably, the oligonucleotide of the invention targets an mRNA encoding Translationally-Controlled Tumor Protein (TCTP), and is capable of reducing the amount of TCTP in cells.

Nucleic acids that target an mRNA encoding TCTP may be designed by using the sequence of said mRNA as a basis, e.g. using bioinformatic tools. For example, the sequence of SEQ ID NO: 5 can be used as a basis for designing nucleic acids that target an mRNA encoding TCTP.

Preferably, the oligonucleotides according to the invention are capable of reducing the amount of TCTP in cells, e.g. in cancerous cells such as LNCaP or PC3 cells. Methods for determining whether an oligonucleotide is capable of reducing the amount of TCTP in cells are known to the skilled in the art. This may for example be done by analyzing TCTP protein expression by Western blot, and by comparing TCTP protein expression in the presence and in the absence of the oligonucleotide to be tested (see Figure 8 and Example 4).

The oligonucleotides according to the invention typically have a length of from 12 to 50 nucleotides, e.g. 12 to 35 nucleotides, from 12 to 30, from 12 to 25, from 12 to 22, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 22, from 18 to 22, or about 19, 20 or 21 nucleotides.

The oligonucleotides according to the invention may for example comprise or consist of 12 to 50 consecutive nucleotides, e.g. 12 to 35, from 12 to 30, from 12 to 25, from 12 to 22, from 15 to 35, from 15 to 30, from 15 to 25, from 15 to 22, from 18 to 22, or about 19, 20 or 21 consecutive nucleotides of a sequence complementary to the mRNA of SEQ ID NO: 5.

In particular, the inventors have identified thirteen oligonucleotides targeting an mRNA encoding TCTP that are very efficient in reducing the amount of TCTP in cells. These oligonucleotides target the regions consisting of nucleotides 153 to 173 of SEQ ID NO: 5, nucleotides 220 to 240 of SEQ ID NO: 5, nucleotides 300 to 320 of SEQ ID NO: 5, and nucleotides 320 to 340 of SEQ ID NO: 5, respectively. All of these oligonucleotides target the translated region of the TCTP mRNA (which extends from nucleotide 94 to 612 of SEQ ID NO: 5).

Therefore, the oligonucleotides according to the invention preferably target a sequence overlapping with nucleotides 153 to 173, or with nucleotides 221 to 240 or with nucleotides 300 to 340 of SEQ ID NO: 5, said oligonucleotide being a DNA or a RNA. Such an oligonucleotide may for example target: - a sequence consisting of nucleotides 153 to 173 or of nucleotides 221 to 240 or of nucleotides 300 to 320, or of nucleotides 320 to 340 of SEQ ID NO: 5, or

- a sequence comprised within nucleotides 153 to 173 or within nucleotides 221 to 240, or within nucleotides 300 to 320, or within nucleotides 320 to 340 of SEQ ID NO: 5, or

- a sequence partially comprised within nucleotides 153 to 173 or within nucleotides 221 to 240, or within nucleotides 300 to 320, or within nucleotides 320 to 340 of SEQ ID NO: 5.

The oligonucleotides according to the invention may for example comprise a fragment of at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 2 (5’-ACCAAT G AGOG AGT CAT CAA-3’) , SEQ ID NO: 3 (5’-AACCCGUCCGCGAUCUCCCGG-3’), SEQ ID NO: 6 (5’-AACTTGTTTCC TGCAGGTGA-3’), SEQ ID NO: 7 (5’-TGGTTCATGACAATATCGAC-3’), SEQ ID NO: 8 (5’-TAATCATGATGGCGACTGAA -3’), SEQ ID NO: 16 (5’-ACCAGTGATTA CTGTGCTTT-3’), SEQ ID NO: 17 (5’-CTTGTAGGCTTCTTTTGTGA-3’), SEQ ID NO: 18 (5’-ATGTAATCTTTGATGTACTT-3’), SEQ ID NO: 19 (5’-GTTTCCCTTTG ATTGATTTC-3’), SEQ ID NO: 20 (5’-TTCTGGTCTCTGTTCTTCAA-3’), SEQ ID NO: 25 (5’- AG AAAAT CAT AT ATGGGGT C -3’), SEQ ID NO: 27 (5’-TTAACATTTCTCC ATTTCTA -3’), SEQ ID NO: 29 (5’-GT CAT AAAAGGTTTT ACT CT-3’) and SEQ ID NO: 31 (5’-GAAATTAGCAAGGATGTGCT-3’). More preferably, the oligonucleotides comprise a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31 . The oligonucleotides according to the invention may for example comprise a fragment of at least 10 consecutive nucleotides of a sequence of SEQ ID NO: 2 or of a sequence of SEQ ID NO: 6, or of a sequence of SEQ ID NO: 7, or of a sequence of SEQ ID NO: 3. Most preferably, they comprise a sequence of SEQ ID NO: 2, or a sequence of SEQ ID NO: 6, or a sequence of SEQ ID NO: 7 or a sequence of SEQ ID NO: 3. Still preferably, the oligonucleotides according to the invention comprise a fragment of at least 10 consecutive nucleotides of a sequence of SEQ ID NO: 6. Most preferably, they comprise a sequence of SEQ ID NO: 6.

In a preferred embodiment according to the invention, the oligonucleotide is an antisense oligonucleotide. As used herein, the term "antisense oligonucleotide" refers to a single stranded DNA or RNA with complementary sequence to its target mRNA, and which binds its target mRNA thereby preventing protein translation either by steric hindrance of the ribosomal machinery or induction of mRNA degradation by ribonuclease H.

The antisense oligonucleotide may be a DNA or a RNA molecule.

Said antisense oligonucleotide may for example comprise or consist of a fragment of at least 10, 12, 15, 18 or 20 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO: 2 (5’-ACCAATGAGCGAGTCATC AA-3’), SEQ ID NO: 6 (5’-AACTTGTTTCCTGCAGGTGA-3’), SEQ ID NO: 7 (5’-TGG TTCATGACAATATCGAC-3’), SEQ ID NO: 8 (5’-TAATCATGATGGCGACTGAA-3’), SEQ ID NO: 16 (5’-ACCAGTGATTACTGTGCTTT-3’), SEQ ID NO: 17 (5’-CTTGT AGGCTTCTTTTGTGA-3’), SEQ ID NO: 18 (5’-ATGTAATCTTTGATGTACTT-3’), SEQ ID NO: 19 (5’-GTTTCCCTTTGATTGATTTC-3’), SEQ ID NO: 20 (5’-TTCTG GTCTCTGTTCTT C A A- 3’ ) , SEQ ID NO: 25 (5’- AG AAAAT CAT AT AT GGGGT C -3’), SEQ ID NO: 27 ( 5’ -TT AAC ATTT CT CC ATTT CT A- 3’ ) , SEQ ID NO: 29 (5’-GTCATAA AAGGTTTT ACT CT-3’) and SEQ ID NO: 31 (5’-GAAATTAGCAAGGATGTGCT-3’), preferably of a sequence SEQ ID NO: 2 (5’-ACCAATGAGCGAGTCATCAA-3’), or of a sequence of SEQ ID NO: 6 (5’- AACTT GTTT COT GCAGGT G A-3’) , or of a sequence of SEQ ID NO: 7 (5’-T GGTT CAT GACAAT AT CG AC-3’) . Preferably, it comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 31 , more preferably, from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 7. Most preferably, it comprises or consists of the sequence SEQ ID NO: 6.

In another preferred embodiment according to the invention, the oligonucleotide is an interfering RNA (iRNA).

RNA interference is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et ai, 1998, Nature 391 :806-81 1 ). dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNA interference involves mRNA degradation, but many of the biochemical mechanisms underlying this interference are unknown. The use of RNA interference has been further described in Carthew et al. (2001 , Current Opinions in Cell Biology, 13:244- 248) and in Elbashir et al. (2001 , Nature, 41 1 :494-498). The iRNA molecules of the invention are double-stranded or single-stranded RNA, preferably of from about 21 to about 23 nucleotides, which mediate RNA inhibition. That is, the iRNA of the present invention preferably mediate degradation of mRNA encoding TCTP.

The term“iRNA” include double-stranded RNA, single-stranded RNA, isolated RNA (partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA), as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). Nucleotides in the iRNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. Collectively, all such altered iRNA compounds are referred to as analogs or analogs of naturally-occurring RNA. iRNA of the present invention need only be sufficiently similar to natural RNA that it has the ability to mediate RNA interference. As used herein the phrase "mediate RNA Interference" refers to and indicates the ability to distinguish which mRNA are to be affected by the RNA interference machinery or process. RNA that mediates RNA interference interacts with the RNA interference machinery such that it directs the machinery to degrade particular mRNAs or to otherwise reduce the expression of the target protein. In one embodiment, the present invention relates to iRNA molecules that direct cleavage of specific mRNA to which their sequence corresponds. It is not necessary that there be perfect correspondence of the sequences, but the correspondence must be sufficient to enable the iRNA to direct RNA interference inhibition by cleavage or lack of expression of the target mRNA.

The iRNA molecules of the present invention may comprise an RNA portion and some additional portion, for example a deoxyribonucleotide portion. The total number of nucleotides in the RNA molecule is suitably less than 49 in order to be effective mediators of RNA interference. In preferred RNA molecules, the number of nucleotides is 16 to 29, more preferably 18 to 23, and most preferably 21 -23.

As indicated above, the term“iRNA” includes but is not limited to siRNAs, shRNAs, miRNAs, dsRNAs, and other RNA species that can be cleaved in vivo to form siRNAs.

A “short interfering RNA” or “siRNA” comprises a RNA duplex (double- stranded region) and can further comprise one or two single-stranded overhangs, 3’ or 5’ overhangs. A “short hairpin RNA (shRNA)” refers to a segment of RNA that is complementary to a portion of a target gene (complementary to one or more transcripts of a target gene), and has a stem-loop (hairpin) structure.

“MicroRNAs” or“miRNAs” are endogenously encoded RNAs that are about 22-nucleotide-long, that post-transcriptionally regulate target genes and are generally expressed in a highly tissue-specific or developmental-stage-specific fashion. One can design and express artificial miRNAs based on the features of existing miRNA genes. The miR-30 (microRNA 30) architecture can be used to express miRNAs (or siRNAs) from RNA polymerase II promoter-based expression plasmids (Zeng et al, 2005, Methods enzymol. 392:371 -380). In some instances the precursor miRNA molecules may include more than one stem-loop structure. The multiple stem-loop structures may be linked to one another through a linker, such as, for example, a nucleic acid linker, a miRNA flanking sequence, other molecules, or some combination thereof.

In a preferred embodiment according to the invention, the iRNA comprises or consists of a fragment of at least 10, 12, 15 or 18 consecutive nucleotides of a sequence of SEQ ID NO: 3 (5’-AACCCGUCCGCGAUCUCCCGG-3’). Preferably, such a nucleic acid is an iRNA comprising or consisting of a sequence of SEQ ID NO: 3. Most preferably, such a nucleic acid is a siRNA or a shRNA. The sequence of SEQ ID NO: 3 may be modified, and e.g. correspond to a modified sequence of SEQ ID NO: 3 such as a 5’-AACCCGUCCGCGAUCUCCCdGdG-3’ sequence.

The present inventors also demonstrated that modified oligonucleotides comprising an oligonucleotide consisting of an adenine 15-mer of sequence SEQ ID NO: 32 (dAi 5 ) or of a thymidine 15-mer of sequence SEQ ID NO: 33 (dTi 5 ) were particularly useful to form micelles capable of encapsulating phenazine and phenazine derivatives.

Accordingly, in a preferred embodiment, the oligonucleotide is selected from the group consisting of dTi 5 , dAi 5 , and an oligonucleotide consisting of the sequence SEQ ID NO: 6.

Lipid conjugate

According to an embodiment, the oligonucleotide, as defined in the section " Oligonucleotide " herein above, is modified by substitution at the 3’ or the 5’ end by a moiety comprising at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chains comprising from 1 to 22 carbon atoms, preferably from 6 to 20 carbon atoms, in particular 10 to 19 carbon atoms, and even more preferably from 12 to 18 carbon atoms.

According to another embodiment, the oligonucleotide, as defined in the section " Oligonucleotide " herein above, is modified by substitution at the 3’ or the 5’ end by a moiety comprising at least three saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chains comprising from 2 to 30 carbon atoms, preferably from 5 to 20 carbon atoms, more preferably from 10 to 18 carbon atoms.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the general formula (I):

Oligo

wherein:

• Oligo represents an oligonucleotide sequence which may be oriented 3’-5’ or 5’-3’, simple and/or double stranded, ADN, ARN, and/or comprise modified nucleotides, in particular an oligonucleotide as defined in the section " Oligonucleotide " herein above;

• X represents a divalent linker moiety selected from -O- (ether), -S- (thio), -NH- (amino) and -CH 2 - (methylene);

• Ri and R 2 may be identical or different and represent:

(i) a hydrogen atom,

(ii) a halogen atom, in particular fluorine atom,

(iii) a hydroxyl group,

(iv) an alkyl group comprising from 1 to 12 carbon atoms;

• Li and l_ 2 may be identical or different and represent a saturated or unsaturated, linear or branched hydrocarbon chain comprising from 1 to 22 carbon atoms,

• B is an optionally substituted nucleobase, selected from the group consisting of purine nucleobases, pyrimidine nucleobases, and non-natural monocyclic or bicyclic heterocyclic nucleobases wherein each cycle comprises from 4 to 7 atoms.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the general formula (II):

Oligo

wherein:

• Oligo represents an oligonucleotide sequence which may be oriented 3’-5’ or 5’-3’, simple and/or double stranded, ADN, ARN, and/or comprise modified nucleotides, in particular an oligonucleotide as defined in the section " Oligonucleotide " herein above;

• X represents a divalent linker moiety selected from -O- (ether), -S- (thio), -NH- (amino) and -CH 2 - (methylene);

• Ri and R 2 may be identical or different and represent:

(i) a hydrogen atom,

(ii) a halogen atom, in particular fluorine atom,

(iii) a hydroxyl group,

(iv) an alkyl group comprising from 1 to 12 carbon atoms;

• Mi, M 2 and M 3 may be identical or different and represent:

- a saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chain comprising from 2 to 30 carbon atoms, preferably from 6 to 22 carbon atoms, more preferably from 12 to 20 carbon atoms, which may be substituted by one or more halogen atoms, notably be fluorinated or perfluorinated and/or be interrupted by one or more groups selected from ether -0-, thio -S-, amino -NH-, oxycarbonyl -O-C(O)-, thiocarbamate -0-C(S)-NH-, carbonate -0-C(0)-0-, carbamate -0-C(0)-NH-, phosphate -0-P(0)(0)-0- and phosphonate -P-0(0)(0)- groups; and/or be substituted at the terminal carbon atom by an aliphatic or aromatic, notably benzylic or naphtylic ester or ether group;

- an acyl radical with 2 to 30 carbon atoms, preferably with 6 to 22 carbon atoms, more preferably with 12 to 20 carbon atoms, or

- an acylglycerol, sphingosine or ceramide group. In the context of the invention, the term "alkyl" refers to a hydrocarbon chain that may be a linear or branched chain, containing the indicated number of carbon atoms. For examples, C1-C12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it.

In the context of the invention, the term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl or heteroarylcarbonyl substituent.

Preferably, the oligonucleotide sequence“Oligo-” is connected to the divalent linker moiety X via a phosphate moiety -0-P(=0)(0 )-, at its 3' or 5' end, advantageously at its 5’ end.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the general formula (I’):

wherein:

- X, R1, R2, Li, l_ 2 and B are as defined above in formula (I),

- [3’— -5’] represents, along with the P0 3 residue, an oligonucleotide as defined in the section " Oligonucleotide " herein above, and

- A + represents a cation, preferably H + , Na + , K + or NH 4 + .

In the formulae (I) and (I’), the divalent linker moiety is preferably -O- (ether).

In the formulae (I) and (I’), R1 and R 2 are preferably hydrogen atoms.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the formula (I”):

wherein A + , X, Li, l_ 2 and B are as defined above in formula (I) and [3’— -5’] represents, along with the P0 3 residue, an oligonucleotide as defined in the section " Oligonucleotide " herein above.

In the formulae (I), (G) and (I”), L and l_ 2 preferably represent a hydrocarbon chain, preferably a linear hydrocarbon chain, comprising from 6 to 22 carbon atoms, preferably from 8 to 18 carbon atoms, advantageously from 12 to 16 carbon atoms, more advantageously 15 carbon atoms.

In the formulae (I), (G) and (I”), B preferably represents a non substituted nucleobase selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 6-methoxypurine, 7-methylguanine, xanthine, 5,6-dihydrouracil, 5- methylcytosine, 5-hydroxymethylcytosine and hypoxanthine. Preferably, in the formulae (I), (G) and (I"), B represents a non substituted nucleobase selected from the group consisting of uracil, thymine, adenine, cytosine, 6-methoxypurine and hypoxanthine. More preferably, in the formulae (I), (G) and (I"), B represents uracil.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the formula (G”):

wherein A + is as defined above in formula (I) and [3’— -5’] represents, along with the P0 3 residue, an oligonucleotide as defined in the section " Oligonucleotide " herein above. In a preferred embodiment according to the invention, the modified oligonucleotide is of the general formula (II’):

wherein:

- X, Ri , R 2 , MI , M 2 and M 3 are as defined above in formula (II),

- [3’— -5’] represents, along with the P0 3 residue, an oligonucleotide as defined in the section " Oligonucleotide " herein above, and

- A + represents a cation, preferably H + , Na + , K + or NH 4 + .

In the formulae (II) and (II’), the divalent linker moiety is preferably ether -0-.

In the formulae (II) and (IG), Ri and R 2 are preferably hydrogen atoms.

In a preferred embodiment according to the invention, the modified oligonucleotide is of the formula (II”):

wherein A + , Mi , M 2 and M 3 are as defined above in formula (II) and [3’— -5’] represents, along with the P0 3 residue, an oligonucleotide as defined in the section " Oligonucleotide " herein above.

In the formulae (II), (II’) and (II”), Mi , M 2 and M 3 preferably represent a hydrocarbon chain, preferably a linear hydrocarbon chain, comprising from 6 to 22 carbon atoms, preferably from 12 to 20 carbon atoms, more preferably 18 carbon atoms. In a preferred embodiment according to the invention, the modified oligonucleotide is of the fo

wherein A + is as defined above in formula (I) and [3’— -5’] represents, along with the P0 3 residue, an oligonucleotide as defined in the section " Oligonucleotide " herein above.

In formula (II’”), the chains -C I 8 H 3 7 are preferably straight alkyl chains.

The modified oligonucleotide may be prepared by a process comprising the following steps:

(i) synthesizing the oligonucleotide, as defined in the section " Oligonucleotide " herein above;

(ii) modifying the oligonucleotide by reaction with a suitable reactant comprising a ketal functional group comprising two saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chains comprising from 1 to 22 carbon atoms, preferably from 6 to 20 carbon atoms, more preferably from 12 to 18 carbon atoms;

(iii) recovering the modified oligonucleotide.

Such process is described in the international application WO 2014/195430.

The modified oligonucleotide may be prepared by a process comprising the following steps:

(i) synthesizing the oligonucleotide, as defined in the section " Oligonucleotide " herein above;

(ii) modifying the oligonucleotide by reaction with a suitable reactant comprising a moiety having at least three saturated or unsaturated, preferably saturated, linear or branched, preferably linear, hydrocarbon chains comprising from 2 to 30 carbon atoms, preferably from 6 to 22 carbon atoms, more preferably from 12 to 20 carbon atoms;

(iii) recovering the modified oligonucleotide.

Such process is described in the international application WO 2014/195432. Active compound

The nanoparticle of the invention comprises at least one active compound selected from phenazine and derivatives thereof. Said active compound is thus loaded or encapsulated into said nanoparticle (or nanocapsule).

As mentioned above, said active compound is loaded into the core of the nanoparticle (or in the micelle formed by the modified oligonucleotide).

Phenazine derivatives are known as useful in the treatment of pancreatic and prostate cancers, as described in WO 201 1/1 17830.

According to an embodiment, the active compound is a phenazine derivative having the following formula

wherein Ri, R 2 , R3 and R 4 , independently from each other, are selected from the group consisting of:

- a hydrogen atom;

- a halogen atom chosen among chlorine, fluorine, bromine and iodine atom;

- a cyano group;

- -NO2;

- a -N(R 5 )(R 6 ) group, with R 5 and R 6 representing, independently the one of the other, a hydrogen atom, a linear or branched (Ci-C 2 o)alkyl group, a benzyl group or a -COR10 group;

- an amido group;

- a -NHS0 2 (R 7 ) group, R ? representing a linear or branched (Ci-C 20 )alkyl group or an aryl group;

- a -ORs group, R 8 representing H or a linear or branched (Ci-C 20 )alkyl group;

- a -OCORg group, R 9 representing H or a linear or branched (Ci-C 20 )alkyl group;

- a (CrC 2 o)alkyl or (Ci-C 20 )alkylthio group, linear or branched, saturated or non saturated,

- a -COR10 group, R10 representing a hydroxyl group or a linear or branched (CrC 2 o)alkoxy group, and

- a -CO-[NH-(CH 2 )n]m-N(R 5 )(R 6 ), -HN 0-[(CH 2 ) n -N(Rii)]p-(CH 2 )n-N(Rii)(R 6 ), or -0-C0-[(CH 2 ) n -N(Rii)]p-(CH 2 )n-N(Rii)(R 6 ) group, . R 5 and R 6 being are such as defined above,

. R representing a hydrogen atom or a tert-butyloxycarbonyl group,

. p representing an integer ranging from 0 to 1 , and

. n, n' and m, independently, representing an integer with n and n', independently, ranging from 2 to 6 and m ranging from 1 to 2, and, when m is 2, each n value may be identical or different one from another, or anyone of its pharmaceutically acceptable salts. In formula (III), the term“alkyl” means a saturated aliphatic hydrocarbon group which may be straight or branched. Preferred alkyl groups have 1 to 20 carbon atoms in the chain. Preferred alkyl groups are in particular methyl, ethyl, propyl or dodecyl groups. "Branched" means that one or lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain.

The term“alkoxy” refers to a -O-alkyl group, with the alkyl group being as defined above. Among alkoxy, it may be cited, the methoxy, ethoxy, propoxy or isopropoxy groups.

The term“alkylthio” refers to a -S-alkyl group, with the alkyl group being as defined above.

The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system comprising from 6 to 10 carbon atoms wherein any ring atom capable of substitution may be substituted by a substituent. In one embodiment, aryl groups are not substituted. Examples of aryl moieties include, but are not limited to phenyl or naphtyl.

The term“halogen” refers to the atoms of the group 17 of the periodic table and includes in particular fluorine, chlorine, bromine, and iodine atoms, more preferably fluorine, chlorine and bromine atoms.

The process of preparation of the compounds of general formula (III) is described in WO 201 1/1 17830, more specifically in page 13 and in pages 16 to 20 of WO 201 1/1 17830. In one embodiment, the active compound is a derivative of formula (III) wherein:

- R I =R 2 and represent R 8 , -OR 8 or -OCORg, with R 8 representing a linear or branched (CrC 2 o)alkyl group, and Rg representing a hydrogen atom or a linear or branched (Ci-C 20 )alkyl group;

- R 3 and R 4 , independently of each other, represent:

- a hydrogen atom,

- a linear or branched (Ci-C 20 )alkyl,

- a linear or branched (CrC 20 )alkenyl,

- -N(R 5 )(R 6 ), with R 5 and R 6 representing independently of each other: a hydrogen atom, a linear or branched (Ci-C 20 )alkyl group, a benzyl group or a -COR10 group, Rio being a hydroxyl group or a linear or branched (CrC 20 )alkoxy group;

- -CO-[NH-(CH 2 )n]m-N(R 5 )(R 6 ), -HN-CO-[(CH 2 )n-N(Rii)]p-(CH 2 )n-N(Rii)(R 6 ), or - 0-C0-[(CH 2 )n-N(Rii)]p-(CH 2 )n'N(Ri i)(R 6 ) with:

- R 5 and R 6 being as defined above,

- R1 1 representing a hydrogen atom or a tert-butyloxycarbonyl group,

- p representing an integer ranging from 0 to 1 , and

- n, n' and m, independently of each other, representing an integer with n and n', independently, ranging from 2 to 6 and m ranging from 1 to 2, and, when m is 2, each n value may be identical or different one from another, or one of its pharmaceutically acceptable salts.

In one embodiment, in the compound of the formula (III), both Ri and R 2 are a linear or branched -O(Ci-C 20 )alkyl group.

In another embodiment, in the compound of the formula (III), R3 and R 4 represent independently of each other: a hydrogen atom, -N(R 5 )(R6) or -CO-[NH- (CH 2 )n]m-N(R 5 )(R6), with n, m, R 5 and R 6 being as above. In a particular embodiment, R 5 and R 6 are H or methyl, ethyl or propyl. Preferably n is 2 and/or m is 1 . In one embodiment, the compound of formula (III) has one of the following formulae:

The compounds of general formula (III) herein described may have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well-known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a compound are intended, unless the stereochemistry or the isomeric form is specifically. The term "pharmaceutically acceptable salt" refers to salts which retain the biological effectiveness and properties of the compounds of the invention and which are not biologically or otherwise undesirable.

The compounds of general formula (III) may be provided in the form of a free base or in the form of addition salts with acids. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. For a review of pharmaceutically acceptable salts see Berge, et al. ((1977) J. Pharm. Sd, vol. 66, 1 ). For example, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, fumaric, methanesulfonic, and toluenesulfonic acid and the like.

According to a most preferred embodiment, in the nanoparticle according to the invention, the active compound has the following formula: Method of preparation of the nanoparticles according to the invention

The nanoparticles according to the invention may be prepared according to known techniques. The nanoparticles according to the invention may be prepared by nanoprecipitation or by formation of a lipid film.

In one embodiment, a process of preparation of the nanoparticles according to the invention comprises the following steps:

i) solubilizing an active compound, preferably having formula (III) as defined above, in an organic solvent, such as chloroform, thereby obtaining a mixture (M);

ii) removing the organic solvent from the mixture (M), thereby obtaining a hydrophobic film; and

iii) rehydrating the hydrophobic film with an oligonucleotide solution, said oligonucleotide being as defined above.

Preferably, the mixture (M) is evaporated under inter gas with stirring in step ii). According to an embodiment, the organic solvent may further be removed under high vacuum.

According to an embodiment, in step iii), the oligonucleotide solution is added dropwise under fast stirring.

To improve the homogeneity of the nanoparticular suspension, an ultrason bath (15 min) may be perfomed twice.

The solution obtained after step iii) may then be filtered to remove the insoluble large aggregates of non-encapsulated active compound.

Composition

The present invention also provides an aqueous composition comprising modified oligonucleotides as defined hereabove, wherein the modified oligonucleotides self-assemble into aggregates, wherein at least one active compound as defined above is loaded.

A micelle is an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in aqueous solution forms an aggregate with the hydrophilic "head" regions of the molecules in contact with surrounding aqueous solvent, sequestering the hydrophobic“tail” regions of the molecules in the micelle center.

The modified oligonucleotides self-assemble into aggregates having a core/shell structure, wherein the shell is hydrophilic and is formed of the oligonucleotide parts of the modified oligonucleotides, and wherein the core is lipophilic and is formed of the saturated or unsaturated, linear or branched, hydrocarbon chains in C1-C22 of the modified oligonucleotides.

The aqueous compositions may comprise up to 50% by weight of modified oligonucleotides, preferably from 0.1% to 40%, in particular from 1% to 20%, and especially from 8% to 15% by weight of modified oligonucleotides.

As mentioned above, the aqueous composition of the invention further comprises an active compound hosted in said aggregates as defined above.

Loading of such aggregates with an active compound can vary between 2 mM to 2 mM.

Therefore, the aqueous composition as defined above is used as a carrier or vehicle of the active compound as defined above. Such a vehicle is notably useful for the administration of such active substance by way of intravenous, intraperitoneal, subcutaneous or oral routes, or direct hemoral injection.

Uses of the nanoparticles according to the invention

The invention also relates to the nanoparticles as defined above, for use as a medicament.

In a particular embodiment, the nanoparticles of the invention are intended for use in the prevention and/or treatment of cancers.

The invention also relates to a method of prevention and/or treatment of cancers, comprising the administration to a mammal, preferably a human, in need thereof of a therapeutically effective amount of the nanoparticles according to the invention.

“Cancer” means the uncontrolled, abnormal growth of cells and includes within its scope all the well-known diseases that are caused by the uncontrolled and abnormal growth of cells as well as metastasis.

In one embodiment, the cancer is chosen among the group consisting of lymphomas, angiosarcomas and the cancers of the lung, pancreas, breast, bladder, colon, skin, head and neck, ovarian, and prostate.

In a particular embodiment, the cancer is selected from the group consisting of: prostate cancer, breast cancer and pancreatic cancer, preferably prostate cancer such as resistant prostate cancer. In a particular embodiment, the cancer is the hormono-resistant prostate cancer (also called castration resistant prostate cancer). By“resistant prostate cancer” it may be meant a prostate cancer for which hormonotherapy and/or chemotherapy is(are) not sufficient to cure said prostate cancer. In particular, “resistant prostate cancer” is a prostate cancer for which hormonotherapy and/or chemotherapy do(es) not allow to:

- inhibit the tumor growth (tumor stasis); and/or

- decrease partially the tumor; and/or

- suppress the tumor.

In one embodiment, “resistant prostate cancer” may refer to: metastatic prostate cancers; prostate cancers which cannot be treated by surgery or radiation; and prostate cancers which remain or come back after treatment with surgery or radiation therapy.

Hormonotherapy is also called androgen deprivation therapy (ADT) or androgen suppression therapy. The goal of this treatment is to reduce levels of androgens (testosterone and dihydrotestosterone) in patients, or to stop them from affecting prostate cancer cells. In hormonotherapy, LHRH agonists, LHRH antagonists, CYP17 inhibitors, anti-androgens, or estrogens may be used.

Chemotherapy is a category of cancer treatment that uses chemical substances which are anti-cancer cells agents. In particular, docetaxel, cabazitaxel, mitoxantrone, or estramustine may be used in the treatment of prostaste cancer.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. In particular, the treatment of cancers may consist in destroying and/or depleting cancer cells and/or preventing resistance and/or restore sensitivity to hormonotherapy and/or chemotherapy of cancer cells.

According to the invention, the term "patient" or "individual" to be treated is preferably intended for a human or non-human mammal (such as a rodent, for example a mouse or a rat, a feline, a canine, or a primate) affected or likely to be affected with a disease. Preferably, the patient is a human.

A "therapeutically effective amount" or "therapeutic dose" is an amount sufficient to obtain the desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose can be administered in one or more administrations. In particular, a therapeutically effective dose is an amount that is sufficient to treat the disease as defined above, in particular cancers as defined above. The invention also relates to a pharmaceutical composition, preferably an aqueous pharmaceutical composition, comprising the nanoparticles as defined above and optionally one or more pharmaceutically acceptable excipient(s).

The present invention also relates to a drug, comprising the nanoparticles as defined above.

While it is possible for the nanoparticles of the invention to be administered alone, it is preferred to present them as pharmaceutical compositions. The pharmaceutical compositions, both for veterinary and for human use, useful according to the present invention comprise at least one nanoparticle as above defined, together with one or more pharmaceutically acceptable carriers and possibly other therapeutic ingredients.

In certain embodiments, active ingredients necessary in combination therapy may be combined in a single pharmaceutical composition for simultaneous administration.

As used herein, the term "pharmaceutically acceptable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

The preparation of a pharmacological composition that contains the nanoparticles as defined above dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified. In particular, the pharmaceutical compositions may be formulated in solid dosage form, for example capsules, tablets, pills, powders, dragees or granules.

The choice of vehicle is generally determined in accordance with the physical and chemical properties of the nanoparticles, the particular mode of administration and the provisions to be observed in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulphate and talc may be used for preparing tablets. To prepare a capsule, it is advantageous to use lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.

The pharmaceutical compositions can be administered in a suitable formulation to humans and animals by topical or systemic administration, including oral, rectal, nasal, buccal, ocular, sublingual, transdermal, rectal, topical, vaginal, parenteral (including subcutaneous, intra-arterial, intramuscular, intravenous, intradermal, intrathecal and epidural), intracisternal and intraperitoneal. In a particular embodiment, the pharmaceutical composition as defined above is administered by intravenous injection.

It will be appreciated that the preferred route may vary with for example the condition of the recipient.

The formulations can be prepared in unit dosage form by any of the methods well known in the art of pharmacy. In general the formulations are prepared by uniformly and intimately bringing into association the nanoparticles with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 shows the sequence of human TCTP protein.

SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 to SEQ ID NO: 31 show ASO sequences.

SEQ ID NO: 3 shows the sequence of a TCTP siRNA according to the invention.

SEQ ID NO: 4 shows the sequence of a scramble oligonucleotide that has been used as a control.

SEQ ID NO: 5 shows the sequence of a human TCTP mRNA. The regions that are targeted by the TCTP ASO of SEQ ID NO: 2 and by the TCTP siRNA of SEQ ID NO: 3 are indicated.

SEQ ID NO: 32 shows the sequence of dAi 5 .

SEQ ID NO: 33 shows the sequence of dTi 5 . FIGURES

Figure 1 shows the percentage of cell viability at several concentrations (in mM) for non-encapsulated phenazine, DMSO, and nanoparticles according to the invention.

Figure 2 shows the percentage of cell viability for control, DMSO, LASO, non- encapsulated phenazine (Ph14), nanoparticles according to the invention (nanocompund LASO-Ph14), and LASO+Ph14.

Figure 3 shows the percentage of cell viability (normalized to NT) for ASO- Phenazine.

The invention will now be described more in detail by way of the examples below and the drawings in annex.

EXAMPLES

Preparation of ASO-Phenazine and LASO-Phenazine

200 pL of a phenazine stock solution (Tetrahedron Letters Volume 56, Issue 21 , 20 May 2015, Pages 2695-2698, https://doi.Org/10.1016/j.tetlet.2015.04.003) at 5 mg/mL dissolved in chloroform (Sigma) was added into a glass tube and then evaporated under inert gas with stirring to form a hydrophobic film. The residual organic solvent was removed under high vacuum. Then, the hydrophobic film was rehydrated with 1 mL of the oligonucleotide solutions (LASO or ASO, at a concentration of 170 mM dissolved in distilled water) which were added drop wise under fast stirring.

To improve the homogeneity of the nanoparticular suspension an ultrasonic bath (15 min) was perfomed twice. This solution was filtered through a 1 pm by syringe filter to remove the insoluble large aggregates of non-encapsulated phenazine. This formulation (N/P = 1 ) was characterized by DLS, TEM and Electrophoretic Light Scattering.

The quantification of phenazine and oligonucleotide in the nanoparticles was performed after destabilization of the nanoparticles. Thus, a saturated sodium chloride solution was used to disrupt the phenazine/oligo complexes and a liquid/liquid extraction with chloroform and 0.9% NaCI were performed. The aqueous phase was extracted 5 times with chloroform to separate oligonucleotides and phenazine which were dissolved into the aqueous and organic phases, respectively. Both phases were evaporated under reduce pressure with rotary concentrator (RVC 2-18 CDplus - Christ) and dissolved into distilled water (oligonucleotides) or chloroform (phenazine) in final volume of 600 mI_. Phenazine was dosed by spectrophotometry at 425 nm ( = 0,O0725lS2x + O,0D898361) The oligonucleotide concentration was measured by spectrophotometry at 260 nm with the Beer-Lambert law and molar extinction coefficient of oligonucleotide (determined by OligoAnalyser tools software IDT DNA).

Confocol microscopy

Intracellular distribution of Phenazine and LASO-Phenazine was analyzed by confocal microscopy. One day before transfection, PC-3 cells (8 c 10 4 cells/well) were plated on 8-well Lab-Tek II chamber slides (Nunc-Thermo Scientific). Cells were then transfected with auto-fluorescent Phenazine or LASO-Phenazine; cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 min at 4°C, and mounted in Prolong Gold mounting medium containing the nuclear counterstain DAPI (Life technologies). For visualization, we used a Zeiss LSM 510 META fluorescence microscope with 350-nm and 450-nm excitation filters.

Cell viability assay

Cell viability was determined by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium (MTT) assay. PC-3 cells were plated in 12-well plates (3 c 10 4 cells/well) and transfected the day after with increasing concentrations of Phenazine, DMSO or TCTP-Phenazine. After 48 h, MTT was added to each well (1 mg/ml final concentration) and the plates were incubated for 2 h at 37°C. Supernatants were then removed and formazan crystals were dissolved in DMSO. The absorbance (595 nm) was evaluated using a Sunrise microplate absorbance reader (Tecan). Cell viability was expressed as the percentage of absorbance of transfected cells compared to untreated cells.

Results

The main problem of Phenazine 14 is its poor solubility in biological fluids. The inventors were able to encapsulate phenazine 14 in TCTP-LASO. Here, it is shown that phenazine 14 encapsulation in LASO nanocomposite, leads to a better solubility and distribution of the compound.

To evaluate the effect of LASO-Phenazine, a cell viability test was performed on PC-3 cells.

Interestingly, it has been shown (Figure 2) that at 50 mM, LASO-Phenazine nanocomposite induces a 50% decrease in cell viability, while at this concentration the compound alone had no effect.

To evaluate whether the synergic effect is due to the nanoencapsulation of Phenazine in LASO micelle, the experiment was repeated, with cells treated with

DMSO, LASO, Phenazine and TCTP-Phenazine alone or LASO+ Phenazine (Figure 3). Cell viability was expressed as the percentage of absorbance of transfected cells normalized to untreated cells. In this experiment, results were even better, and equivalent concentrations of phenazine 14 (50 mM), and TCTP-LASO (32 mM) led to complete cell death of the culture when encapsulated, while the effects of phenazine

14 added to TCTP-LASO were lower.

In order to check whether Phenazine encapsulation is optimized by LASO micelles, we generated an ASO-Phenazine solution, and we checked its effect on cell viability as described before (Figure 4). Unlike LASO-Phenazine, no effect was observed for ASO-Phenazine at all doses tested.