EP2945935A1

EP2945935A1 - Amphiphilic derivatives of triazamacrocyclic compounds, products and composition including same, and synthesis methods and uses thereof

Info

Publication number: EP2945935A1
Application number: EP14704380.6A
Authority: EP
Inventors: Stéphane MOUTARD; Vincent DELAUZUN; Laurent Meunier
Original assignee: Biocellchallenge
Current assignee: Biocellchallenge
Priority date: 2013-01-21
Filing date: 2014-01-21
Publication date: 2015-11-25
Also published as: CA2898081A1; FR3001217A1; FR3001217B1; US9822083B2; WO2014111639A1; US20150353514A1

Abstract

The invention relates to amphiphilic derivatives of a triazamacrocyclic compound, as well as to said derivatives as active molecule transporters. The invention also relates to a nanodrug including at least one amphiphilic derivative of a triazamacrocyclic compound and at least one active molecule of a protein such as an antibody, in particular for the treatment of autoimmune diseases or for the treatment of cancer.

Description

AMPHIPHILIC DERIVATIVES OF TRIAZAMACROCYCLE COMPOUNDS, PRODUCTS AND COMPOSITIONS COMPRISING THE SAME, AND METHODS THEREOF

OF SYNTHESIS AND USES THEREOF

The present invention relates to a novel family of lipids in the field of drug chemistry. More particularly, the invention relates to amphiphilic derivatives of triazamacrocycle compounds for the intracellular transport of molecules of therapeutic interest, in particular for the intracellular delivery of antibodies.

To date, there is a strong demand for intracellular delivery systems for molecules with therapeutic potential. Genome sequencing and the very rapid development of genomics since the mid-1990s has led to the discovery and development of many delivery systems dedicated to nucleic acids. However, research studies and the discovery of intracellular delivery systems adapted to other types of potentially therapeutic molecules, including antibodies, are still quite rare.

Unlike the intracellular delivery of genes for which many viral approaches go beyond biochemical or physical approaches to deliver a transgene into a cell, and integrate it into its genetic heritage, the transport of other types of molecules can only be realized with physical and biochemical approaches.

The physical approaches make use, for example, of electroporation, microinjection or sonoporation techniques. They make it possible to deliver nucleic acids or other types of molecules inside the cells. These techniques are used with more or less success in vitro and are in most cases difficult to apply in vivo.

Through a biochemical approach, ^■ cationic lipids and cationic polymers are used, inter alia, as carriers. The most efficient molecules have many positive charges, which interact electrostatically with nucleic acids, with many negative charges.

The complexes formed between the nucleic acids and cationic molecules are:

polyplexes when the complexes are formed with a cationic polymer, or

lipoplexes when the complexes are formed with a cationic lipid.

In the case of lipoplexes, the nucleic acids are encapsulated in vesicular structures called liposomes.

Lipoplexes or polyplexes interact with cell membranes and then deliver their contents inside the cells according to their own mechanisms. However, if this approach is rather effective to deliver nucleic acids in the cell, at least in vitro, given the well-defined physico-chemical characteristics of nucleic acids, it is quite different for other types of molecules. For example, if the overall negative charge of the nucleic acids allows for complexation with cationic transporters, the complexation of proteins with such transporters is much more uncertain. In this approach, it appears that the encapsulation of the molecule to be transported in liposomes is the most appropriate. To date, liposomes are the most used tools for the intracellular delivery of genes or molecules therapeutic. Hydrophilic or lipophilic molecules may be encapsulated in these liposomes. These liposomes may consist of several lipids but in the majority of currently used formulations, they contain at least one cationic lipid. The cationic lipid is generally associated with a neutral α-lipid such as DOPE (1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine). Thus, a large number of cationic lipids have been developed, generally for the transport of genes in cells.

The first lipid developed for gene transport in cells is DOTMA as reported in Felgner et al., Proc. Nat. Acad. Sci. 84, 7413 (1987). DOTMA is N- (1- (2,3-dioleyloxy) propyl) -N, -trimethylammonium chloride.

The chemical formula of DOTMA is given below:

Other molecules have also been developed ranging from rather simple structures such as DOTAP (1, 2-dioleoyl-3-trimethylammonium-propane) of formula:

or the DMRIE (1, 2-DiMyristyl Rosenthal Inhibitor Ether) formula:

Molecules of rather complex structures to see much more complex have also been developed. he is for example DOGS (Dioctadecylamidoglycyl permine) of formula:

or similar lipid membrane lipids of archaea ^1:

However, the above lipid molecules have been selected and investigated for their ability to carry nucleic acids in cells for gene therapy purposes. Some of the synthesized molecules were then used to deliver into the cells molecules other than nucleic acids such as proteins. However, these systems have been inefficient and unreliable until now.

With regard to protein transport in cells, scientists generally employ sophisticated, time-consuming, costly and limited methods in their applications. The most studied approach is to use a special class of peptides called protein transduction domains (PTDs). The mechanism of action of PTDs is not defined to date, and their delivery efficiency varies according to the protein transported. In addition, one of the major disadvantages of using these PTDs is the need to chemically couple them to the protein to be transported for use.

There is therefore a need to date to develop new carriers or vectors capable of efficiently and reliably transporting molecules that are not only nucleic acids, such as for example proteins. Such carriers or vectors should ideally allow to implement simple methods, fast and low cost. They should also be able to have a wide spectrum of applications.

The solution to the problem posed relates to an amphiphilic derivative of triazamacrocycle compound of formula (I):

(I)

in which :

R ¹ corresponds to formula (II):

(E) q - (T) r - (L ^ sd ^ t

(II)

and wherein:

E represents a hydrocarbon group, linear or branched, saturated or unsaturated, comprising from 1 to 15 carbon atoms and optionally comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine; T represents a branched hydrocarbon group, saturated or unsaturated, comprising from 1 to 15 carbon atoms and optionally containing one or more heteroatoms ^■ selected from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine;

L ¹ and L ² , which are identical or different, represent a linear, branched and / or cyclic, saturated or unsaturated hydrocarbon-based group comprising from 6 to 24 carbon atoms and optionally comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine;

q is an integer equal to 0 or 1;

r is an integer equal to 0 or 1;

- s and t, identical or different, are integers equal to 0 or 1, provided that at least one of these integers is different from

⁰ '

R ² represents a hydrogen atom or a linear hydrocarbon group, branched and / or cyclic, saturated or unsaturated, comprising from 1 to 20 carbon atoms, comprising one or more heteroatoms chosen from nitrogen, oxygen and sulfur; fluorine, chlorine, bromine or iodine; and having at least one cationic functional group;

R ³ represents a hydrogen atom or is identical to

R ¹ , or else is identical to R ² .

m, n and p, same or different are integers equal to 0, 1, 2, 3 or 4.

Surprisingly, the Applicant has been able to demonstrate, as reported in the examples, that the amphiphilic derivatives according to the invention are easy to synthesize and allow the efficient transport of active molecules or proteins such as antibodies, even preserved in the presence of bovine serum albumin. In addition, the Applicant has been able to demonstrate that the amphiphilic derivatives according to the invention are effective in different cell types.

The subject of the present invention is also the use of a derivative according to the invention as an intracellular transporter of active molecules.

Its third object is a pharmaceutical composition comprising at least one derivative according to the invention and a pharmaceutically acceptable carrier.

Its fourth object is a nanomedicine comprising, on the one hand, derivatives as described above and, on the other hand, at least one active molecule, that is to say having a therapeutic interest.

A nanomedicine can be defined as a carrier or nanoscale vector capable of bringing an active molecule onto a given therapeutic target: a gene, a protein, a cell, a tissue or an organ.

The invention also has for its fifth object a nanomedicine for use in the treatment of autoimmune diseases, cancers, neurodegenerative pathologies, certain infectious diseases or pathologies of genetic origin resulting in a specific dysfunction in the body.

It also has for its sixth object a product containing a nanomedicine according to the invention and at least a second active compound as a combination product for simultaneous administration, separated or spread over time, for the transport of active molecules of therapeutic interest, in particular for intracellular delivery of antibodies.

Finally, its last object is a process for synthesizing the derivatives according to the invention. The various compounds previously described can in particular be synthesized according to the methods described in Example 1 below, which describe the synthesis of cationic lipids based on TACN (triazacyclononane) for delivering proteins and especially antibodies in living cells.

Among other possible applications, the use of the derivatives according to the invention also makes it possible to study the function of the molecules transported or to block or induce an intracellular function in living cells. By way of example, the transport of monoclonal antibodies into cells can be used to specifically block an intracellular target. This approach has already been demonstrated via the transfection of DNA encoding antibodies called "Intrabodies". Similarly, by the use of the derivatives according to the invention, it becomes possible to use therapeutic antibodies within the cells themselves whereas, until now, the therapeutic antibodies all have targets outside the cells. alive.

The invention will be better understood on reading the nonlimiting description which follows, written with reference to the appended drawings, in which:

Figure 1 illustrates the reaction scheme for the synthesis of 1,4-didodecyl-7-betainyl-1,4,7-triazacyclononane;

FIG. 2 illustrates the reaction scheme for the synthesis of 1,4-dioctadecenyl-7-betainyl-1,4,7-triazacyclononane;

Figure 3 illustrates the reaction scheme for the synthesis of 1,5-dioleyl- (7'-carboxymethyl-1 ',', 7'-triazacyclononane) -L-glutamate; Figure 4 illustrates the reaction scheme for the synthesis of 1,5-dioleyl- (1 ·, 4'-dibetainyl-1 *, 4 ', 7'-triazacyclononane-7'-carboxymethyl) -L-glutamate;

- Figure 5 shows the influence of the amount of DOPE on the effectiveness of the formulation of the compound;

Figures 6a and 6b illustrate the transport of fluorescent antibodies in NIH3T3 cells at 24 h (6a) and 48 h (6b);

Figures 7a and 7b illustrate the transport of fluorescent antibodies in A549 (7a) and RAW264 (7b) cells at 5h.

Figure 8 shows the evaluation of the cellular toxicity of the compound on HeLa cells for 48 hours; Figure 9 shows the kinetics of antibody transport on NIH3T3 cells;

Figure 10 illustrates the influence of PBS on the rapidity of antibody transport in VERO cells;

FIGS. 11a and 11b show the influence of the ionic strength of the medium on the efficiency of the transport

(IIa) and the amount of antibody transported (11b) in A549 and BEAS-2B cells;

FIG. 12 represents the influence of the amount of antibody on the transport efficiency in NIH3T3 cells;

FIG. 13 illustrates, in three different views, the transport of an anti-NFkB p50 antibody in A549 cells;

FIG. 14 represents the transport of an antibody in the mouse; and

FIGS. 15a and 15b show that it is possible to inhibit the growth of tumor cells by means of compounds complexed with an antibody directed against an intracellular protein involved in cell proliferation and oncogenesis. The compounds according to the invention are amphiphilic derivatives of triazamacrocycle compounds of formula (I):

⁽ I)

in which :

R ¹ corresponds to formula (II):

(E) q - (T) r - (L ^ stL ^ t

(II)

and wherein:

E represents a hydrocarbon group, linear or branched, saturated or unsaturated, comprising from 1 to 15 carbon atoms and optionally comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine;

T represents a branched hydrocarbon group, saturated or unsaturated, comprising from 1 to 15 carbon atoms and optionally comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or bromine; iodine;

L ¹ and L ² , which are identical or different, represent a linear, branched and / or cyclic, saturated or unsaturated hydrocarbon-based group comprising from 6 to 24 carbon atoms and optionally comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine; q is an integer equal to 0 or 1;

r is an integer equal to 0 or 1;

s and t, which are identical or different, are integers equal to 0 or 1, provided that at least one of these integers is different from 0;

R ³ represents a hydrogen atom or is identical to

R ¹ , or else is identical to R ² .

m, n and p, same or different are integers equal to 0, 1, 2, 3 or 4.

In what precedes and what follows, the term "heteroatoms", an atom selected from nitrogen, oxygen, sulfur and halogens such as fluorine, chlorine, bromine or iodine. Azamacrocycle is understood to mean a cyclic macromolecule containing one or more nitrogen atoms as represented in formula (I).

Preferably, in formula (II):

(E) q - (T) r - (L ^ sd ^ t

(II)

E, which serves as a spacer arm, corresponds to the following formula (III):

-X-GL-

(III)

in which :

X represents a bridging alkylene group having 1 to 8 carbon atoms; - G1 represents a group -CO-, -O-, -S-, -NH- or -NR- in which R is an alkyl group, advantageously Ci-C.

More preferably, X is a bridging alkylene group having 1, 2, 3 or 4 carbon atoms. Even more preferably, X represents a bridging alkylene group comprising only one carbon atom.

Preferably, in formula (II):

(E) q - (T) r - (L ^ sd ^ t

(II)

T, which serves as a branched spacer arm, represents either the residue of an amino acid or the remainder of a glycerol.

The term "amino acid residue" means the group of atoms that remains of this amino acid when it is covalently bound:

on the one hand on the spacer arm E in the formula (II) or directly on one of the nitrogen atoms of the azamacrocycle in the formula (I), and

- on the other hand, to one and / or the other group L ¹ and

L ² in formula (II).

The term "glycerol residue" means the group of atoms that remains of this glycerol when it is covalently bound:

on the other hand, to one and / or the other of the groups L ¹ and L ² in the formula (II).

Preferably, when T represents the residue of an amino acid in formula (II), T is chosen from the twenty amino acids which conventionally belong to the constitution of proteins, namely aspartic acid, glutamic acid, alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, Isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine.

More preferably, it is selected from aspartic acid, glutamic acid, leucine, isoleucine and lysine, aspartic acid and glutamic acid.

Alternatively, this amino acid may be chosen from among rarer amino acids, for example β-alanine, γ-aminobutyric acid, α-aminoadipic acid, 1-hydroxylysine, acid, ε-aminobutyric acid, diaminopimelic, α, β-diaminopropionic acid, γ-diaminobutyric acid, and ornithine.

In general, any amino acid may be suitable insofar as the amino acids contain, by definition, at least two functional groups, one carboxylic acid, the other amino, allowing its covalent bond:

More preferably, T is the residue of an amino acid belonging to the L series. However, it is also possible that T is the residue of a D-series amino acid.

Preferably, in formula (II):

(E) q - (T) r - (L ^ siL ^ t

(II)

L ¹ and / or L ² have the following formula (IV):

(IV)

in which : G 2 represents a group -CO-, -O-, -S-, -NH- or -NR-, where R is an alkyl group, advantageously a Ci to Ce, and

Y represents a saturated or unsaturated Cs to C24 linear alkyl chain. Y may also represent a cyclic or polycyclic group known to be lipophilic such as a steroid group, for example derived from cholesterol, a polyaromatic group, for example derived from naphthalene, dansyl, anthracene, or a group derived from alkaloids. .

More preferably, Y is a linear saturated or unsaturated alkyl chain of C _i2 to C _8.

More preferably still, the derivative of formula (I) according to the invention is such that:

R ¹ corresponds to formula (II):

(E) q- (T) r- (L ^ siL ^ t

(II)

in which :

E corresponds to the following formula (III):

-X-Gi- (III)

in which :

X represents a bridging alkylene group and comprising from 1 to 8 carbon atoms;

- G is -CO-, -0-, -S-, -NH- or -NR- wherein R is alkyl to C _6;

T represents either the residue of an amino acid or the remainder of a glycerol;

L ¹ and / or L ² correspond to the following formula (IV):

in which : G 2 represents a group -CO-, -O-, -S-, -NH- or -NR-, where R is a C 1 -C 6 alkyl group, and

Y represents a saturated or unsaturated Cs to C24 linear alkyl chain or a cyclic or polycyclic group.

Among the derivatives of triazamacrocycle compounds according to the invention, those in which the spacer arm E is linked by an amide bond or an ester bond to the branched spacer arm T are preferred, T being itself bound by an amide bond or an ester bond. group (s) L ¹ and / or L ² . This for reasons of ease of preparation.

In this case, E preferably corresponds to the formulas -X-CO- or -X-NH- in which X has the same meaning as above. L ¹ and / or L ² preferably correspond to the formulas -OY, -CO-Y or -NH-Y in which Y has the same meaning as above.

In this case also, it is preferred that R ¹ corresponds to formula (V):

in which :

• X and Y have the same meaning as before

• Z represents:

a covalent bond, in which case R ⁴ represents a hydrogen atom, a methyl group, the side chain of an amino acid, or a group of formula (VI):

- (CH2) k-CO-O-Y

(VI) _, in which k is 1 or 2, and Y has the same meaning as before,

is a hydrocarbon-forming group comprising 1, 2, 3 or 4 carbon atoms, and may comprise one or more heteroatoms chosen from O and N, in which case R ⁴ represents a primary amine group or a group of formula (VII):

-NH-CO-Y

(VII)

in which Y has the same meaning as before.

More preferably, the derivative of formula (I) according to the invention is such that:

R ¹ corresponds to formula (V):

(V)

in which :

X represents a bridging alkylene group and having 1, 2, 3 or 4 carbon atoms;

Y represents a saturated or unsaturated C12 to C18 linear alkyl chain;

Z represents:

(VI)

wherein k is 1 or 2, and Y is a saturated linear or unsaturated C12 to

Cis, is a hydrocarbon-forming group comprising 1 to 4 carbon atoms, and may comprise one or more heteroatoms chosen from 0 and N, in which case R ⁴ represents a primary amine group or a group of formula (VII):

-NH-CO-Y

(VII)

wherein Y is a linear saturated or unsaturated alkyl chain of C12 to C _8;

R ² represents a hydrogen atom or a linear hydrocarbon group, branched and / or cyclic, saturated or unsaturated, comprising from 1 to 20 carbon atoms, comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine; and having at least one cationic functional group; and

• R ³ represents a hydrogen atom or is identical to R ¹ , or else is identical to R ² .

Advantageously, when T represents in formula (II):

(E) q- (T) r- (L ^ siL ^ t

(II)

the remainder of an amino acid selected from aspartic acid and glutamic acid, then, in formula (V):

(V)

Z represents a covalent bond;

Y preferably represents a saturated or unsaturated linear alkyl chain C 8 to C 18 and more preferably C 12 to C 18; R 5 represents a group of formula (VI): embedded image in which k is equal to 1 or 2, and Y represents, preferably, a linear saturated or unsaturated C ₈ alkyl chain; to Cis and, more preferably, Cl ₂ to ^_'8 Ci.

According to yet another preferred embodiment of the invention, in formula (I):

(I)

R ² represents a hydrogen atom or the remainder of a cationic amino acid. In the latter case, the term "residue of a cationic amino acid", the group of atoms that subsists an amino acid having at least one cationic functional group when it is covalently bound to one of the atoms nitrogen of the azamacrocycle in the formula (I). Preferably, the cationic functional group (s) carried by this cationic amino acid are amino, guanidino, imidazole or quaternary ammonium groups. In particular, they are chosen from ornithine, lysine, arginine and glycine betaine, glycine betaine being particularly preferred.

In formula (I), R ² preferably represents a hydrogen atom.

According to yet another preferred embodiment of the invention, in formula (I), R ³ represents more particularly a hydrogen atom.

Preferably, the compounds according to the invention are amphiphilic derivatives of triazamacrocycle compounds for which m, n and p are integers equal to 2. Said preferred derivatives are triazacyclononane derivatives which correspond to formula (VIII):

(VIII)

wherein R ¹ , R ² and R ³ are as defined above.

Even more particularly, the preferred derivatives corresponding to formula (VIII) are such that:

R ¹ corresponds to formula (V):

(V)

in which :

^♦ X represents an alkylene bridging group and having 1, 2, 3 or 4 carbon atoms;

· Y is a linear saturated or unsaturated alkyl chain -C ₂ to C _8;

• Z represents:

- (CH2) k-CO-O-Y

(VI)

wherein k is 1 or 2, and Y is a saturated linear or unsaturated C12 to

CL8 is a hydrocarbon-forming group comprising 1 to 4 carbon atoms, and may comprise one or more heteroatoms chosen from 0 and N, in which case R ⁴ represents a primary amine group or a group of formula (VII):

-NH-CO-Y

(VII)

wherein Y represents a saturated or unsaturated C 2 to C 18 linear alkyl chain;

R ³ represents a hydrogen atom or is identical to

R ¹ , or else is identical to R ² .

Among the amphiphilic derivatives of triazamacrocycle compounds according to the invention, it is particularly preferred:

1,4-didodecyl-7-betainyl-1,4,7-triazacyclononane;

1,4-ditetradecyl-7-betainyl-1,4,7-triazacyclononane;

β1,4-dihexadecyl-7-betainyl-1,4,7-triazacyclononane;

1-dioctadecyl-7-betainyl-1,4,7-triazacyclononane;

1,4-dioleyl-7-betainyl-1,4,7-triazacyclononane;

1,4-Didodecyl-N (7'-carboxymethyl-1 ', 4', 7'-triazacyclononane) -L-aspartate;

• the 1, 5-didodecyl-N (7-carboxymethyl-1 ', 4', 7 ¹ -triaza- cyclononane) -L-glutamate; 1,4-ditetradecyl-N (7'-carboxymethyl-1 ', 4', 7'-triazacyclononane) -L-aspartate;

1, 5-ditetradecyl- (7 ^{1-carboxymethyl-l} ^1, 4 ', 7 ¹ -tri- azacyclononane) -L-glutamate;

1,4-dihexadecyl-N (7'-carboxymethyl- ¹ , 4 ', 7'-triazacyclononane) -L-aspartate;

1, 5-dihexadecyl-N (7 ^{1-carboxymethyl-1} ', 4', 7 '-tri- azacyclononane) -L-glutamate;

1, 4-dioctadecyl-N (7-carboxymethyl-1 ', 4', 7 ¹ -tri- azacyclononane) -L-aspartate;

1, 5-dioctadecyl (7 ^{1-carboxymethyl-1} ', 4', 7 '-tri- azacyclononane) -L-glutamate;

1,4-dioleyl- (7'-carboxymethyl- ¹ , 4 ', 7'-triazacyclononane) -L-aspartate;

1,5-dioleyl-N (7'-carboxymethyl- ¹ , 4 ', 7 ^1- triazacyclononane) -L-glutamate;

1, 4-dioleyl-N (1 ', 4' -dibetainyl-7 '-carboxymethyl- 1', 4 ', 7 ¹ triaza-cyclononane) -L-aspartate;

1, 5-dioleyl (1 ^1, 4 '-dibetainyl-7 ¹ -carboxymethyl- 1', 4 ', 7' triaza-cyclononane) -L-glutamate;

1,4-dioctadecyl-7-ornithyl-1,4,7-triazacyclononane;

1,4-dioleyl-7-ornithyl-1,4,7-triazacyclononane; 1,4-dioctadecyl-7-arginyl-1,4,7-triazacyclononane;

1,4-dioleyl-7-arginyl-1,4,7-triazacyclononane; 1,4-dioctadecyl-7-lysyl-1,4,7-triazacyclononane; 1,4-dioleyl-7-lysyl-1,4,7-triazacyclononane;

1,4-dioleyl-N (1 ', 4'-ornithyl-1', 4 ', 7'-triazacyclononon-7'-carboxymethyl) -L-aspartate;

1,5-dioleyl (1 ', 1'-ornithyl-1', 4 ', 7'-triazacyclononon-7'-carboxymethyl) -L-glutamate; • 1, 4-dioleyl-N (1 ', ¹ -arginyl-1', 4 ', 7 ¹ -triaza- cyclononane-7 carboxymethyl) -L-aspartate; and

1,5-dioleyl-N (1 ', 4'-arginyl-1', 4 ', 7'-triazacyclononane-7'-carboxymethyl) -L-glutamate;

• 1, 4-dioleyl-N (1 ', ¹ -diornithyl 4-7 ¹ -carboxymethyl- 1', 4 ', 7' -triazacyclononane) -L-aspartate;

• the 1, 5-dioleyl-N (1 ^1, 4 '-diornithyl-7' -carboxymethyl-

I ', 4 ^1, 7' -triazacyclononane) -L-glutamate;

• 1, 4-dioleyl-N (1 ^1, 4 '-diarginyl-7 ¹ -carboxymethyl-

I ^I, 4 ', 7' -triazacyclononane) -L-aspartate;

1,5-dioleyl-N (1,4'-dienginyl-7'-carboxymethyl-1 ', 4', 7'-triazacyclononane) -L-glutamate

1-octadecyl-4-oleyl-7-betainyl-1,4,7-triazacyclononane;

1-octadecyl-5-oleyl-N (7'-carboxymethyl-1 ', 4,7'-triaza-cyclononane) -L-glutamate;

• 1-octadecyl, oleyl 4-N ^{(1-carboxymethyl-1} 7 4 ', 7' - triaza-cyclononane) -L-aspartate;

1-dodecyl-4-oleyl-7-betainyl-1,75-triazacyclononane;

1-oleyl-4-dodecyl-7-betainyl-1,4,7-triazacyclononane.

Advantageously, the derivatives according to the invention may comprise groups making it possible to increase their solubility, their cellular penetration and their bioavailability.

Another subject of the invention relates to the use of a derivative as described above, as carrier of active molecules.

The active molecules that can be used include any drug, that is to say any substance that can be used in humans or animals or that can be administered to them, with a view to establishing a medical diagnosis. or to restore, correct or modify their physiological functions by exerting a pharmacological, immunological or metabolic action.

By way of nonlimiting examples of active molecules that may be used, mention may be made in particular of proteins such as antibodies, nucleic acids such as genes or siRNAs, but also antitumor agents such as bortezomib, cisplatin, carboplatin, ifosfamide, chlorambucil, busulfan, thiotepa, 5-fluorouracil (5FU), fludarabine, methotrexate, vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, camptothecin derivatives, amsacrine,. anthracyclines, epipodophyllotoxin derivatives From 1 ^1, doxorubicin, daunorubicin, actinomycin D, mitomycin C, plicamycin and bleomycin. ITK can also be used in various tumor diseases such as Imatinib, Dasatinib, Nilotinib and Sunitinib.

Advantageously, the object of the invention derivatives can be used as transporter of nucleic acids or therapeutic molecules such as proteins. More preferably, the derivatives which are the subject of the invention are used as antibody transporters.

The invention further relates to a pharmaceutical composition comprising an amphiphilic derivative as described above.

Preferably, said composition comprises: a derivative as described above;

a co-lipid; and

a pharmaceutically acceptable carrier.

By way of nonlimiting example of co-lipid, mention may advantageously be made of dioleylphosphatidylethanolamine (DOPE). The concentration of the co-lipid in the composition is advantageously between G and 50% by weight of the total weight of the composition. More preferably, the concentration of the co-lipid is between 0 and 30% by weight of the total weight of the composition. More preferably still, the concentration of the co-lipid is between 5 and 10% by weight of the total weight of the composition.

The composition is advantageously in the form of an aqueous dispersion of nanoparticles.

The subject of the invention is also a product comprising, on the one hand, at least one derivative as described above and, on the other hand, at least one active molecule as described above, as a medicament. This type of product may also be named nanomedicine. As indicated above, a nanomedicine can be defined as a nano-sized carrier or vector capable of bringing an active molecule onto a given therapeutic target: a gene, a protein, a cell, a tissue or an organ.

Thus, the invention also relates to a nanomedicine comprising on the one hand, at least one derivative as described above and on the other hand, at least one active molecule as described above.

The invention also relates to such a nanomedicine for the treatment of autoimmune diseases, or for the treatment of cancer.

The invention also relates to a product containing on the one hand a nanomedicine and on the other hand, at least one pharmaceutical compound, as a combination product for simultaneous administration, separated or spread over time, for the transport of molecules of interest. therapeutic, including intracellular delivery of antibodies. Advantageously, the pharmaceutical compound according to the invention is an anti-inflammatory agent, or an agent decreasing the side effects related to nanomedicines or active molecules according to the invention.

By simultaneous therapeutic use, within the meaning of the present invention, is meant an administration of both the nanomedicine and at least one pharmaceutical compound, by the same route and at the same time or substantially at the same time.

By separate therapeutic use, within the meaning of the present invention, is meant in particular an administration of the nanomedicine according to the invention and a pharmaceutical compound, at the same time or substantially at the same time by different routes.

By therapeutic use spread over time means administration of the nanomedicine according to the invention and a pharmaceutical compound at different times, the administration route being identical or different. More particularly, it is intended to mean a mode of administration according to which the entire administration of the nanomedicine according to the invention or of a pharmaceutical compound is carried out before the administration of the other (s) begins.

It is thus possible to administer the nanomedicine according to the invention or a pharmaceutical compound for several months before administering the other. There is no simultaneous treatment in this case. It is also possible to envisage alternating administration of the nanomedicine according to the invention or of the pharmaceutical compound for several weeks.

The route of administration of the composition according to the invention may be orally, parenterally, topically or ocularly. Preferably, the pharmaceutical composition is packaged in a form suitable for parenteral application. Thus, parenterally, the composition may be in the form of solutions or suspensions for infusion or for intramuscular, intravenous or subcutaneous injection.

Alternatively, the composition may be administered orally and may be in the form of tablets, capsules, dragees, syrups, suspensions, solutions, powders, granules, emulsions, suspensions of microspheres. or nanospheres or lipid or polymeric vesicles for controlled release.

Still alternatively, when the composition according to the invention is administered topically, the pharmaceutical composition according to the invention is more particularly intended for the treatment of skin and mucous membranes and may be in liquid, pasty or solid form, and more particularly in the form of ointments, aqueous, hydroalcoholic or oily solutions, lotion-type dispersions, aqueous, anhydrous or lipophilic gels, powders, soaked swabs, syndets, wipes, sprays, foams, of sticks, shampoos, compresses, washing bases, emulsions of liquid or semi-liquid consistency of the milk type, obtained by dispersion of a fatty phase in an aqueous phase (O / W) or conversely (W / O) ), or suspensions or emulsions of soft, semi-liquid or solid consistency of the cream, gel or ointment type. It may also be in the form of suspensions of microspheres or nanospheres or lipid or polymeric vesicles or polymeric or gelled patches allowing controlled release.

Finally, the subject of the invention is also processes for synthesizing amphiphilic derivatives according to the invention. Such synthetic methods are described in the examples below. Example 1 Synthesis of compounds according to the invention

1. Material:

The majority of reagents and solvents come from Alfa Aesar GmbH (Bischheim, France), Merck KgaA (Darmstadt, Germany), VR Prolabo (Briare, France), Sigma-Aldrich (Saint Quentin Fallavier, France) and Fluka (division of Sigma-Aldrich, Saint Quentin Fallavier, France). Triazacyclononane (TACN) comes from CheMatech (Dijon, France). All anhydrous solvents are purchased from Merck and used as is.

2. Methods

a) Chromatography

Thin layer chromatography (TLC) is carried out on 5 x 7.5 cm aluminum plates coated with 60 F254 silica gel (Merck). The compounds are revealed under UV light (λ = 254 nm), then by spraying with a 10% aqueous sulfuric acid solution followed by a heating step at 250 ° C. for all the lipid derivatives, or by spraying with water. a 0.2% solution of Ninhydrin in ethanol followed by a heating step at 250 ° C for the compounds having an amine function.

The flash chromatographic separations are carried out on silica gel 60 (230-400 Mesh ASTM) (Merck). b) Mass spectrometry

Sample preparation

The products to be analyzed are dissolved (0.01 mg / mL) in a 1/1 (v / v) methanol / water or acetonitrile / water 1: 1 (v / v) mixture and the solutions are directly introduced into the electrospray source. (at 5 μί / ιηίη) by via a syringe pump (Harvard Apparatus, Les Ulis, France).

Appareiliage

Exact mass measurements are performed on a Waters-Micromass (Manchester, UK) LCT, equipped with a pneumatically assisted electrospray ion source (Z-Spray), and equipped with an additional fogger (Lockspray) for the compound reference (Nal). Nitrogen is used as a desolvation and fogging gas with a flow rate of 500 and 20 L / h, respectively. The temperatures of the source and the desolvation gas are respectively set at 80 and 120 ° C. The capillary voltage is ± 3.0 kV and the cone voltage is ± 100 V (± ESI). The spectra are accumulated at a rate of 3 seconds per scan for a mass range between 100 and 3500 uma. The resolution used is 5000 FWHM.

Data acquisition and processing is done with the MassLynx V3.5 program. c) NMR ^X H

The proton NMR experiments are recorded at a frequency of 400.13 MHz on a BRUKER Avance DRX400 instrument equipped with a Broad Band Inverse (bbi) probe. The chemical shifts are given with respect to an external reference, tetramethylsilane (δ = 0 ppm), and the internal calibrations are carried out using the residual solvent signal. The deuterated solvents employed (CDCl3, DMSO-d6) come from Eurisotop (Gif sur Yvette, France). Measurements are made with rigorous temperature control at 298 K (± 0.1 K).

The spectra are acquired on 16 K points, and transformed on 32 K points (zero-filling). Possible treatment using an exponential function (1 <LB <5) or a Gaussian function (0.1 <GB <0.3; -3 <LB <-1) is performed on each of the spectra, as well as a correction of the baseline.

Spectra are processed on a PC using MestReNova software (Mestrelab Research S.L., Santiago de Compostela, Spain).

3. Examples of synthetic processes

a) Synthesis of 1,4-didodecyl-7-betainyl-1,4,7-triazacyclononane (1) (DL-TACN-Bet)

The synthesis method illustrated in FIG. 1 makes it possible to obtain the title compound, or compound 1, of formula

1

from 1, 4, 7-triazacyclononane commercially i) Step 1: Preparation of 1,4-di (tert-butoxycarbonyl) -1,4,7-triazacyclononane (la)

Triethylamine (460 μL, 3.30 mmol) is added to a solution of 1,78-triazacyclononane (TACN) (320.8). mg; 2.48 mmol) in chloroform (20 mL) with stirring and under an inert atmosphere. A solution of di-tert-butyl dicarbonate (1 g; 4.58 mmol) in chloroform (10 ml) is then added very slowly over a period of 4 hours to the reaction medium. The mixture is then stirred overnight at room temperature and under an inert atmosphere. The solvents are then evaporated to dryness under a primary vacuum, until a white solid residue is obtained, which is purified by chromatography on silica gel (10/1 v / v ethyl acetate / methanol). The pure compound (521.2 mg, 70%) is thus isolated as a colorless oil. TLC: Rf = 0.5 (CH ₂ Cl ₂ / MeOH 9/1 v / v).

HRMS (ESI ⁺ ): m / z measured at 330.2394 [M + H] ⁺ ; calculated at 330.2393 for C ₁₆ H 32 N ₃ O ₄ .

1 H NMR (CDCl ₃ ) δ (ppm): 1.41 (s, 18H, CH ₃ ); 2.85-2.87 (m, 4H, C¾); 3.15 to 3.22 (m, 4H, C¾) ^'; 3.35-3.42 (m, 4H, CH ₂ ). ii) Step 2: Preparation of 3-betainylthiazolidine-2-ione chloride (Ib)

lb

A solution of thionyl chloride (1.8 g, 15 mmol) in anhydrous acetonitrile (20 mL) is added dropwise into the reaction medium containing glycine betaine (1.17 g, 10 mmol) in suspension. in anhydrous acetonitrile (5 mL), glycine betaine having been previously dried at 50 ° C. for 4 days. The The mixture is then maintained at 35-40 ° C. for 1 h with stirring and under an inert atmosphere. The reaction medium is then concentrated under primary vacuum, and the resulting acyl chloride is dissolved in anhydrous dichloromethane (10 mL). 2-mercaptothiozoline (1.31 g, 11 mmol) and triethylamine (1 g, 10 mmol), previously dissolved in 60 mL of anhydrous dichloromethane, are added at 0 ° C in the previous solution. The reaction medium is then stirred for 30 minutes at room temperature and under an inert atmosphere.

After concentration of the medium under a primary vacuum, the precipitate formed is washed twice with boiling dichloromethane and then sintered to give activated glycine 1b (1.66 g, 65%) as a yellow powder.

TLC: Rf = 0.7 (MeOH).

HRMS (ESI ⁺ ): m / z measured at 219.0623 [M] ⁺ ; calculated at 219, 0626 for C ₈ H ₁₅ N ₂ OS ₂ .

^X H NMR (DMSO-d6) δ (ppm): 3.30 (s, 9H _r CH _3); 3.49 (t, 2H, CO-N-C¾, J = 7.6 Hz); 4.57 (t, 2H, CH ₂ -S, J = 7.6 Hz); 5.27 (s, _2H,. N ⁺ -C¾-C0). iii) Step 3: Preparation of 1,4-di (tert-toxycarbonyl) -7-betainyl-1,4,7-triazacyclononane (le)

the

Triethylamine (1 mL, 7.2 mmol) is added to a solution of 1,4-di (tert-butoxycarbonyl) -1,4,7-triazacyclononane la (150 mg, 0.46 mmol) in N, N dimethylformamide (DMF) (10 ml) with stirring and under an inert atmosphere. The 3-betainylthiazolidine-2-thione 1b chloride (175.8 mg, 0.69 mmol) is then added directly to the reaction medium. The mixture is then stirred overnight at room temperature and under an inert atmosphere. The solvents are then evaporated to dryness under a primary vacuum. The residue obtained is finally purified by chromatography on silica gel (ethyl acetate). Compound 1e is thus isolated in the form of a white solid (chloride salt) (117.3 mg, 55%).

TLC: Rf = 0.5 (CH ₂ Cl ₂ / MeOH 9/1 v / v).

HRMS (ESI +): m / z measured at 429, 3074 [M] +; calculated at 429, 3077 for C21H41N4O5. iv) Step 4: Preparation of 7-betainyl-1,4,7-triazacyclononane (

ld

Trifluoroacetic acid (1 mL) is added to a solution of 1,4-di (ert-butoxycarbonyl) -7-betainyl-1,4,7-triazacyclononane (110 mg, 0.24 mmol) in dichloromethane ( 1 mL) with stirring. The reaction is stirred for 30 minutes at room temperature room. The reaction medium is then concentrated under primary vacuum, redissolved in dichloromethane and again concentrated under primary vacuum. The operation is repeated several times to remove any traces of trifluoroacetic acid. The residue is dried overnight under a primary vacuum. Pure compound ld (135 mg, quantitative) is obtained as a pale yellow oil.

TLC: Rf = 0 (CH ₂ Cl ₂ / MeOH 9/1 v / v).

MS (ESI +): m / z measured at 230.2109 [M + H] +; calculated 230.2107 for C _n H ₂₆ N ₄ 0. v) Step 5: Preparation of didodecyl triflate (Ic)

The trifluoromethanesulphonic anhydride (1.8 g, 1.07 mL, 6.5 mmol) and then the anhydrous pyridine (0.51 g, 525 μL, 6.5 mmol) are successively added to a solution of anhydrous dichloromethane (10 mL ) cooled in an ice bath at 0 ° C, with stirring and under an inert atmosphere. During this addition, a release of smoke is observed and a white precipitate is formed in the reaction medium. The ice bath is then removed, then a solution of lauric alcohol (0.93 g, 5 mmol) in anhydrous dichloromethane (4 mL) is added dropwise into the reaction medium with stirring. The mixture is stirred for 2 hours at room temperature, then the reaction is quenched by adding water. Dichloromethane (20 mL) is added to the reaction medium, and the solution is washed with water (2 x 10 mL). The aqueous phases are re-extracted with dichloromethane (2 mL) and the organic phases are pooled, washed with brine (10 mL), dried over sodium sulfate and filtered through filter paper. The filtrate is evaporated in a rotary evaporator to obtain a brown oil. The oil is redissolved in hexane (2 mL) and loaded onto a silica column. The final product is eluted with a 1/1 v / v ether / hexane mixture, being careful not to entrain the colored side products which are co-eluted as a result of the product. The solvents are evaporated to dryness in a rotary evaporator, and the pure product (1.41 g, 89%) is obtained in the form of a colorless oil.

TLC: Rf = 0.7 (Hexane / Ether 1/1 v / v).

R ¾ (CDC1 ₃₎ δ (ppm): 4.56 (t, 2H, CF ₃ S0 ₃ C¾); 1.91-1.79 (m, 2H, CF ₃ SO ₃ CH ₂ C¾);

1, 53-1, (m, 18H, CH 2 (lauryl)); 0.91 (t, 3H, CH ₃ ).

The instability of this product over time requires that it should be used very quickly for the next synthesis step. vi) Step 6: Preparation of 1,4-didodecyl-1-betainyl-1,4,7-triazacyclononane (1)

The diisopropylethylamine (78 μL, 0.45 mmol) is added to a solution of 7-betainyl-1,4,7-triazacyclononane ID (50 mg, 0.09 mmol) in anhydrous chloroform (10 mL) with stirring and under stirring. inert atmosphere. Didodecyltriflate (143.3 mg; 0.45 mmol), freshly prepared from lauryl alcohol and triflic anhydride, is then directly added to the reaction medium. The mixture is then stirred overnight at room temperature and under an inert atmosphere. The solvents are then evaporated to dryness under a primary vacuum. The residue obtained is finally purified by chromatography on silica gel (dichloromethane / methanol 1/0 to 9/1 v / v). The pure compound 1 (41.4 mg, 86%) is thus isolated in the form of a white solid.

TLC: Rf = 0.5 (CH ₂ Cl ₂ / eOH 9/1 v / v).

HRMS (ESI ⁺ ): m / z measured at 566, 5866 [M + H] ⁺ ; calculated at 566.5863 for C35H74N4O. b) Synthesis of 1,4-dioctadecenyl-7-betainyl-1,4,7-triazacyclononane (2) (DO-TACN-Bet)

The synthesis method illustrated in FIG. 2 makes it possible to obtain the title compound, or compound 2, of formula:

This compound 2 is obtained by coupling 7-betainyl-

1, 7-triazacyclononane (1d), the synthesis of which was previously described in point 3a), with oleyl triflate (2a). i) Step 1: Synthesis of oleyl triflate (2a)

The trifluoromethanesulphonic anhydride (1.8 g, 1.07 mL, 6.5 mmol) and then the anhydrous pyridine (0.51 g, 525 μL, 6.5 mmol) are successively added to a solution of anhydrous dichloromethane (10 mL ) cooled in an ice bath at 0 ° C, with stirring and under an inert atmosphere.

During this addition, a release of smoke is observed and a white precipitate is formed in the reaction medium. The ice bath is then removed, and then a solution of oleic alcohol (1.34 g, 5 mmol) in anhydrous dichloromethane (4 mL) is added dropwise into the reaction medium with stirring. The mixture is stirred for 2 hours at room temperature, then the reaction is quenched by adding water. Dichloromethane (20 mL) is added to the reaction medium, and the solution is washed with water (2 x 10 mL). The aqueous phases are re-extracted with dichloromethane (2 ml) and the organic phases are combined, washed with brine (10 ml), dried over sodium sulphate and then filtered on filter paper. The filtrate is evaporated in a rotavapor to obtain a light brown liquid. The liquid residue is redissolved in hexane (2 mL) and loaded onto a silica column. The final product is eluted with a 1/1 v / v ether / hexane mixture, being careful not to entrain the colored side products which are co-eluted as a result of the product. The solvents are evaporated to dryness in a rotary evaporator, and pure product 2a (1.30 g, 65%) is obtained in the form of a colorless liquid.

TLC: Rf = 0.8 (Hexane / Ether 1/1 v / v). ^X H NMR (CDCl3) δ (ppm): 5, 42-5, 33 (m, 2H, CH = CH); 4.54 (t, 2H, CF 3 SO 3 CH 2); 2.13-1.93 (m, 4H, C 1 CH = CHC¾); 1.90-1.69 (m, 2H, CF3 SO3CH2C¾); 1.54-1.12 (m, 22H,

C¾ (oleyl)); 0.89 (t, 3H, C¾).

The instability of this product over time requires that it must be used very quickly for the next synthesis step. II) Step 2: Synthesis of 1,4-dioctadecenyl-7-betalyl-1,4,7-triazacyclononane (2)

Diisopropylethylamine (78 L, 0.45 mmol) is added to a solution of 7-betainyl-1,4,7-triazacyclononane 1d (50 mg, 0.09 mmol) in anhydrous chloroform (10 mL) with stirring and with stirring. inert atmosphere. L ¹ oléyltriflate 2a (180.3 mg, 0.45 mmol), freshly prepared from oleic alcohol and triflic anhydride, is then added directly into the reaction medium. The mixture is then stirred overnight at room temperature and under an inert atmosphere. The solvents are then evaporated to dryness under a primary vacuum. The residue obtained is finally purified by chromatography on silica gel (dichloromethane / methanol 1/0 to 9/1 v / v). The pure compound 2 (53.0 mg, 94%) is thus isolated in the form of an oil.

TLC: Rf = 0.5 (CofC MeOH 9/1 v / v) HR S (ESI +): m / z measured at 730, 7428 [M + H] ₊ ; calculated at 730, 7428 for C47H944O.

c) Synthesis of 1,5-Dioleyl-N (7'-carboxymethyl-1 ', 4', 7'-triazacyclononane) -L-Glutamate (3) (DOG-TACN)

The synthesis method illustrated in FIG. 3 makes it possible to obtain the title compound, or compound 3, of formula:

3

This compound 3 is obtained from 1,4-di (tert-butoxycarbonyl) -1,4-triazacyclononane (1a), the synthesis of which has previously been described in point 3a). i) Step 1: Preparation of 1,4-Di (tert-butoxycarbonyl) -7-carboxymethyl-1,4,7-triazacyclononane (3a)

Sodium carbonate (67 mg, 0.63 mmol) and ethyl bromoacetate (70, 0.63 mmol) are successively added to a solution of 1,4-di (tert-butoxycarbonyl) -1,4,7 -triazacyclononane la (173.6 mg, 0.53 mmol) in acetonitrile (10 mL). The mixture is then refluxed (bath temperature = 80 ° C.) and kept stirring overnight at reflux. Then, the reaction is cooled to room temperature, and the insolubles are removed by filtration. The solution is concentrated under a primary vacuum, and the crude is purified by silica gel chromatography (ethyl acetate) to give the intermediate ester (194 mg, 88%) as a pale yellow oil.

TLC: Rf = 0.8 (CH2C MeOH 9/1 v / v).

HRMS (ESI +): m / z measured at 416, 2758 [M + H] ₊ ; calculated at 416.2761 for CaoHssNaOe.

The ester (194 mg, 0.47 mmol) is dissolved in methanol (5 mL) and then 1 mol / L sodium hydroxide solution (3 mL) is added to the stirred solution. The mixture is stirred for 2 hours at room temperature. The methanol is then evaporated under vacuum, and the pH of the resulting aqueous solution is adjusted to 5 by addition of a solution of citric acid to 5% (m / v). The aqueous phase is then extracted with dichloromethane (3 × 20 ml), the organic phases are combined, dried over anhydrous sodium sulphate, and the filtrate is concentrated under primary vacuum to obtain pure compound 3a (160 mg, 88%). in the form of a colorless oil.

TLC: Rf = 0.4 (CH ₂ Cl ₂ / MeOH 9/1 v / v).

HRMS (ESI +): m / z measured at 388, 2452 [M + H] ₊ ; calculated at 388.2448 for C18H34N3O6.

RN iH (CDCl3) δ (ppm): 1.30-1.50 (m, 18H, C (CH3) 3 (Boc)); _. 3.10-3.60 (m, 14H, C¾ (TACN),

NCH2CO); 8.20 (br s, 1H, OH).

ii) Step 2: Preparation of 1,5-dioleyl-L-glutamate (3b)

3b

L-Glutamic acid (0.5 g, 3.4 mmol) and para-toluenesulfonic acid (0.780 g, 4.1 mmol) are dissolved in toluene (100 mL). The mixture is then refluxed (bath temperature = 120 ° C.) with stirring for 1 h. The oleic alcohol (2.01 g, 7.5 mmol) is then introduced into the reaction medium, then the The reaction is stirred and refluxed overnight. The reaction was then cooled to room temperature, _^ then the toluene is evaporated to dryness under low vacuum. The resulting residue is dissolved in chloroform (50 mL) and washed successively with saturated aqueous sodium bicarbonate solution (2 x 25 mL) and then with brine (1 x 25 mL). The organic phase is dried over anhydrous sodium sulphate, and the filtrate is concentrated under primary vacuum to obtain a crude product which is purified by chromatography on silica gel (dichloromethane / methanol 4/1 to 1/1 v / v). Pure compound 3b (0.45 g, 20%) is thus isolated as a pale yellow oil. CC: Rf = 0.4 (CH 2 Cl 2 / MeOH 98/2 v / v).

HRMS (ESI +): m / z measured at 648, 4931 [M + H] +; calculated at 648.4931 for CIIHÎSNOI.

¹ H NMR (CDCl 3) δ (ppm): 0.90 (t, 6H, CH 2 CH 3 (oleyl)); 1.26 (m, 44H, CH? (Oleyl)); 1.57 (m, 4H, COOCH2CH2 (oleyl)); 1.90-2.12 (m, 2H, NH 2 CHCH 2 (glutamate)); 2.04 (m, 8H, C 1 CH = CHC 2 (oleyl)); 2.47 (t, 2H, CH 2 CO (glutamate)); 3.59 (t, 1H, NH ₂ C (glutamate)); 4.06- 4.15 (t, 4H, COOCl (oleyl)); 5.32-5.42 (m, 4H, CH = CH (oleyl)); 7.18, 7.73 (d, 2H, NH 2). iii) Step 3: Preparation of 1,5-dioleyl-N (1 ', 4'-di (tert-butoxycarbonyl) -7'-carboxymethyl-1', 4 ', 7' -triaza-cyclononan) -L-glutamate (3c)

3c

PyBOP (72 mg, 0.138 mmol), 1,5-Dioctadecenyl-L-Glutamate 3b (97.3 mg, 0.150 mmol) and diisopropylethylamine (66 μL, 0.30 mmol) are successively added to a solution of 1 4-di (tert-butoxycarbonyl) -7-carboxymethyl-1,4,7-triazacyclononane 3a (53.3 mg, 0.138 mmol) in anhydrous dichloromethane

(2 mL) with stirring and under an inert atmosphere. The reaction is then maintained under stirring and under an inert atmosphere overnight at room temperature. The solvents are then evaporated to dryness under a primary vacuum, and the residue is purified by chromatography on silica gel (100/1 v / v dichloromethane / methanol). The pure 3c compound is thus isolated

(124 mg, 88%) as a yellow-orange oil.

TLC: Rf = 0.8 (C Cla / MeOH 95/5 v / v).

HRMS (ESI ₊ ): m / z measured at 1019.8116 [M + H] ₊ ; calculated at 1019.8113 for C59H109N3O10.

NMR Ή (CDCl3) δ (ppm): 0.90 (t, 6H, CH2CH3 (oleyl)); 1.20-1.60 (m, 66H, C (C₁¾) 3 (Boc), C₁¾ (oleyl),

C00CH2C¾ (oleyl)); 1.90-2.12 (m, 2H, N? CHCl? (Glutamate)); . 2.04 (m, 8H, (oleyl)); 2.47 (t, 2H, CH2CO (glutamate)); 3.10-3.60 (m, 15H, Cl- (TACN), NCH2CO, NH ₂ CH (glutamate)); 4.06-4.15 (t, 4H, COOCH2 (oleyl)); 5, 32-5.42 (m, 4H, Ctf = Ctf (oleyl)); 7.29 (d, 1H, NHCO). iv) Step 4: Preparation of 1,5-dioleyl-N (7 '- carboxymethyl-1', 4 ', 7'-triazacyclononane) -glutamate (3) (DOG-TACN)

Trifluoroacetic acid (1 mL) is added to a solution of 1,5-dioctadecenyl- (1 ', 4'-di (tert-butoxycarbonyl) -7 ¹ -carboxymethyl-1', 4 ', 7'-triazacyclononane) -L-Glutamate 3c (124 mg, 0.122 mmol) in dichloromethane (1 mL) with stirring. The reaction is stirred for 30 minutes at room temperature. The reaction medium is then concentrated under primary vacuum, redissolved in dichloromethane and again concentrated under primary vacuum. The operation is repeated several times to remove any traces of trifluoroacetic acid. The residue is dried overnight under a primary vacuum. Pure compound 3 (126 mg, quantitative) is obtained as a pale yellow oil.

TLC: Rf = 0 (CH ₂ Cl ₂ / MeOH 95/5 v / v).

HRMS (ESI ₊ ): m / z measured at 817.7145 [M + H] ₊ ; calculated at 817, 7146 for C49H93N4O5. d) Synthesis of 1,5-dioleyl- (1 ', 4'-dibetainyl-1', 4 ', 7'-triazacyclononane-7'-carboxymethyl) -L-glutamate (DOG-TACN- (Bet) ₂ )

The synthesis method illustrated in FIG. 4 makes it possible to obtain the title compound, or compound 4, of formula:

This compound 4 is obtained by coupling the chloride 3-betainylthiazolidine-2-thione (Ib) with 1,5-dioleyl N (7-carboxymethyl-1 ', 4', 7 ¹ -triazacyclononane) -L-glutamate ( 3), the syntheses of which have previously been described in points 3 (a) and 3 (c), respectively.

Triethylamine (1 mL, 7.2 mmol) is added to a solution of 1,5-dioctadecenyl- (7'-carboxymethyl-1 ', 4,7'-triazacyclononane) -L-glutamate 3 (100 mg, 0.097 mmol) in N, N dimethylformamide (DMF) (10 mL) with stirring and under an inert atmosphere. The 3-betainylthiazolidine-2-thione 1b chloride (74.1 mg, 0.291 mmol) is then added directly to the reaction medium. The mixture is then stirred overnight at room temperature and under an inert atmosphere. The solvents are then evaporated to dryness under a primary vacuum. The residue obtained is finally purified by chromatography on silica gel (dichloromethane / methanol 95/5 to 8/2 v / v). Compound 1e is thus isolated in the form of a white solid (24.2 mg, 23%).

TLC: Rf = 0.4 (CH ₂ Cl ₂ / MeOH 9/1 HRMS (ESI +): m / z measured at 1017, 8672 [M + H] +; calculated at 1017, 8671 _. for CsgHmNeO ?.

The example presented above describes the synthesis of triazacyclononane compounds containing a glycine betaine derivative. Similarly, compounds containing derivatives of arginine, lysine and ornithine have been made. The efficiencies of these compounds for the intracellular transport of antibodies differ depending on the cell type used but they are all more or less active. The presence of triacyclononane is essential to obtain an effective compound.

Example 2: Biological Applications

1. Materials and methods:

Dioleylphosphatidylethanolamine (DOPE) comes from Sigma-Adlrich (Saint Quentin Fallavier, France). The antibodies that it is desired to deliver to the cells are goat serum immunoglobulin G (IgG) from Sigma-Aldrich and mouse anti-NFKB p50 human IgG from BioLegend (San Diego, CA, USA). ). All culture media come from Lonza (Basel, Switzerland).

The transport efficiency of the labeled antibodies in the cells is observed under a fluorescence microscope.

2. Examples of biological applications related to antibody transport in living cells

a) Formulations of amphiphilic derivatives of triazamacrocycle compounds according to the invention in the form of nanoparticles in water.

In a pillbox, one of the amphiphilic derivatives of triazamacrocycle compounds described according to the invention is solubilized in chloroform in the presence of a co-lipid, dioleylphosphatidylethanolamine (DOPE), at different molar ratios. The solvent is then evaporated under a gentle stream of nitrogen and the resulting lipid film is dried under primary vacuum for at least 30 minutes. The lipid film is then rehydrated in sterile deionized water for 2 hours at room temperature. The suspension is finally sonicated using a sonicator equipped with a microprobe (Sonic Ruptor 250, International OMNI, Kennesaw, USA) to obtain an aqueous dispersion of nanoparticles.

The formulations are prepared at a concentration of 1 mmol / L of amphiphilic derivatives of triazamacrocycle compounds in water. In the examples that follow, we will talk about formulation prepared "according to Example 2.2a". b) Influence of DOPE on the effectiveness of the formulation

Several formulations are prepared according to Example 2.2a with different molar ratios of DOPE, namely respectively with 0%, 5%, 10%, 20% and 50% of DOPE. Cos-7 cells are seeded in a 24-well plate (100,000 cells / well) and cultured in 500 μl of DMEM in the presence of serum in a CO 2 incubator for 24 hours. The antibody transport is then carried out as follows:

• 1 g of fluorescein labeled goat serum immunoglobulin G (IgG) is diluted in 10 μl of PBS buffer.

2 μl of formulation prepared according to Example 2.2a are added and the mixture is incubated for 15 minutes at room temperature. • _* 90 μΐ of serum-free DMEM are then added and the final mixture is transferred to Cos-7 cells in culture.

The cells are then incubated in a CO 2 incubator. The transport efficiency is visualized at 24 h and 48 h under fluorescence microscopy after the cells have been fixed with a solution of formalin.

For each formulation tested, the transport efficiency is measured at 5 h by flow cytofluorometry.

The results presented in FIG. 5 show that the maximum efficiency is achieved with the formulation containing 10% of DOPE. c) Transport of a fluorescent antibody in fibroblasts

NIH3T3 cells are seeded in a 24-well plate (100,000 cells / well) and cultured in 500 μl of DMEM in the presence of serum in a CO 2 incubator for 24 hours.

The antibody transport is then carried out as follows:

1 μg of goat serum IgG labeled with fluorescein is diluted in 10 μl of PBS buffer. 2 of the formulation prepared according to Example 2.2a are added and the mixture is incubated for 15 minutes at room temperature.

• 90 x of serum-free DMEM are then added and the final mixture is transferred to the NIH3T3 cells in culture.

• The cells are then incubated in a CO2 incubator. The transport efficiency is visualized at 24 h and 48 h under microscopy at fluorescence after the cells have been fixed with a solution of formalin.

The results presented in FIGS. 6a and 6b show the fibroblasts whose entire cytoplasm has been invaded by the fluorescent antibodies. The labeling is intense, homogeneous and identical at 24 h and 48 h, the cells having continued their cell growth normally. The fibroblast nucleus does not appear to contain antibodies. d) Transport of Fluorescent Antibodies in Other Cell Types

A549 and RAW264 cells are cultured in 24-well plate (100,000 cells / well). Different fluorescent antibodies are then delivered according to the protocol described in Example 2.2b. The cells are observed under a fluorescence microscope after incubation for 5 h.

The results presented in FIGS. 7a and 7b show that the cells have well internalized the different antibodies. e) Evaluation of the cellular toxicity of the compound

HeLa cells are seeded in a 96-well plate (10,000 cells / well) and cultured in 100 L of DMEM in the presence of serum in a CO 2 incubator for 24 hours. The antibody transport is then carried out as follows:

0.3 μg of fluorescein labeled goat serum IgG is diluted in 2.5 μl of PBS buffer. 0.3 μl of formulation prepared according to Example 2.2a is added and the mixture is incubated for 15 minutes at room temperature.

20 μl of DMEM without serum are then added and the final mixture is transferred to the HeLa cells in culture.

• The cells are then incubated in a CO2 incubator. The cellular toxicity of the compound is measured between 5 h and 48 h by an MTT test (3- (4,5-dimethylthiazol-2-yl) bromide).

2,5-diphenyltetrasodium). The 96-well plate is analyzed on a spectrophotometer at 540 nm.

The results presented in FIG. 8 show that the compound is not cytotoxic under these conditions of use. f) Kinetics of the transport of an antibody NIH3T3 cells are seeded in a plate

96 wells (10,000 cells / well) and cultured in 100 μl of DMEM in the presence of serum in a CO 2 incubator for 24 hours. 0.3 μg of fluorescein-labeled goat serum IgG is transported according to the protocol described in Example 2.2e. The cells are then successively washed with PBS and with a trypsin solution, then centrifuged in order to remove the maximum of free antibodies, and are finally fixed with formalin. The intensity of the fluorescence is measured every 2 h, 3 h, 4 h and then every 5 hours by a fluorometer at 525 nm.

The results presented in FIG. 9 show that the maximum amount of antibodies present in the cells is reached in a few hours only. g) Influence of PBS on the transport kinetics of an antibody

VERO cells are seeded in a 24-well plate (100,000 cells / well) and cultured in 500 μl of DMEM in the presence of serum in a CO 2 incubator for 24 hours.

The antibody transport is then carried out as follows:

• 1 μl of fluorescein labeled goat serum IgG is diluted in 10 μl of PBS buffer.

• 1 .mu.l of formulation prepared according to Example 2.2a is added and the mixture is incubated for 15 minutes at room temperature.

190 μl of PBS are then added and the final mixture is transferred directly to the cells.

NIH3T3 in culture.

The cells are then incubated at room temperature for 20 minutes and then the mixture is replaced by DMEM culture medium with serum. The transport efficiency is visualized at 20 minutes under fluorescence microscopy.

The results presented in FIG. 10 show that the antibody is delivered into cultured cells much more rapidly thanks to the action of PBS. h) Influence of the ionic strength of the medium on the effectiveness of the compound

A549 and BEAS-2B cells are seeded in a 24-well plate (100,000 cells / well) and cultured in 500 L of DMEM in the presence of serum in a CO 2 incubator for 24 hours.

The antibody transport is then carried out as follows:

1 μg of fluorescein-labeled goat serum IgG is diluted in 10 μl of PBS buffer or 10 μl of 0.5X PBS buffer or in 10 μl of 0.1X PBS buffer.

2 μl of formulation prepared according to Example 2.2a are added and the mixture is incubated for 15 minutes at room temperature.

90 μM of serum-free DMEM are then added and the final mixture is transferred to the cells in culture. The cells are then incubated in a CO 2 incubator. The transport efficiency is measured at 5 hours by flow cytofluorometry.

The results shown in FIGS. 11a and 11b show that the ionic strength of the medium in which the mixture of the formulation prepared according to Example 2.2a is made with the antibody influences the manner in which the antibody is transported into the cells. The results obtained show the following trend:

The lower the ionic strength of the medium (0.5X PBS and 0.1X PBS), the greater the percentage of cells that internalized the antibody. On the other hand, the amount of antibody present in each cell is inversely proportional. i) Effect of amount of antibody on transport efficiency

NIH3T3 cells are seeded in a 24-well plate (100,000 cells / well) and cultured in 500 μl of DMEM in the presence of serum in a CO 2 incubator for 24 hours. Antibody transport is performed as described in Example 2.2b. The transport efficiency is measured at 5 h by flow cytofluorimetry. The results presented in FIG. 12 show that there is an optimal quantity of antibody required for the same volume of formulation prepared according to the example j) Translocation of the displayed NFkB-p50 protein using an antibody

A549 cells are seeded in a 24-well plate (100,000 cells / well) and cultured in 500 μL of DMEM in the presence of serum in a CO 2 incubator for 24 hours.

The antibody transport is then carried out as follows:

* 1 mouse IgG anti-NFkB p50 human

(containing 1% bovine serum albumin or BSA) labeled with Alexa Fluor 488 is diluted in 10 μl of PBS buffer.

• 90 μΐι of DMEM without serum are then added and the final mixture is transferred to the NIH3T3 cells in culture.

The cells are then incubated in a CO 2 incubator for 4 hours. The cells are then treated with phorbol 12-myristate 13-acetate at 75 ng / mL for 3 h. The transport efficiency of NFkB p50 is visualized under fluorescence microscopy after the cells have been fixed with a solution of formalin.

The results presented in FIG. 13 show a nuclear localization of the NFkBp50 protein.

The labeling is rather punctuate which shows the accumulation of the protein in certain areas of the nucleus under the effect of phorbol ester. k) Use of the compound in in vivo applications

50 μg of goat serum IgG labeled with DightLight 488 are diluted in 400 μl of a solution containing 5% glucose. 6 μΐ; of the formulation prepared according to example 2.2a concentrated ten times are added and the mixture is incubated for 20 minutes at room temperature.

The mixture is then injected into the vein of the tail of an adult mouse. The mouse is sacrificed 2 days later, the tail is removed, milled, homogenized and centrifuged in order to allow the assay of the antibody always present by means of a fluorometer.

The results presented in FIG. 14 clearly show the immunotherapeutic potential of the formulation prepared according to example 2.2a because it is capable of transporting the antibody in the tail of the mouse whereas no fluorescent activity is observed. in the absence of a formulation prepared according to Example 2.2a.

1) Biological activity of an antibody, directed against an oncoprotein, associated with one of the compound prepared as previously described

U87 and 3T6 cells are seeded in a 24-well plate (75,000 cells / well) and cultured in 500 μl of DMEM containing 10% serum in a CO 2 incubator for 16 hours. 1 or 2 μg of a mouse monoclonal antibody directed against an intracellular oncoprotein (anti-tumor antibody) or 1 or 2 μg of a goat serum IgG (control antibody) are transported in the cells according to the protocol described in FIG. Example 2. 2e.

After 48h incubation in a C02 incubator at

37 ° C, the cells are washed with PBS and then peeled off in a trypsin solution before being counted.

The results presented in FIGS. 15a and 15b show that it is possible to inhibit cell proliferation in a specific manner using an antibody. monoclonal directed against an oncoprotein and transported in the cell by a compound synthesized as described in Example 1.

Claims

1. Amphiphilic derivative of triazamacrocycle compound rmule (I):

in which :

• R ¹ corresponds to formula (V):

(V)

in which :

- X represents an alkylene group forming a bridge and

comprising 1, 2, 3 or 4 carbon atoms;

- Y represents a saturated or unsaturated linear alkyl chain in Ci2 to Cie;

- Z represents:

either a covalent bond, in which case R ⁴ represents a hydrogen atom, a methyl group, the side chain of an amino acid, or a group of formula (VI):

(VI) in which k is 1 or 2, and Y represents a linear saturated or unsaturated C12 to Cis alkyl chain,

either a hydrocarbon group forming a bridge, comprising from 1 to 4 carbon atoms, and which may comprise one or more heteroatoms chosen from 0 and N, in which case R ⁴ represents a primary amine group or a group of formula (VII):

-NH-CO-Y

(VII)

in which Y represents a saturated or unsaturated linear C12 to Cie alkyl chain;

• R ² represents a hydrogen atom or a linear, branched and/or cyclic hydrocarbon group, saturated or unsaturated, comprising from 1 to 20 carbon atoms, comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine having at least one cationic functional group chosen from an amino, guanidino, imidazole or quaternary ammonium group;

• R ³ represents a hydrogen atom or is identical to R ¹ , or even is identical to R ²

And

• m, n and p, identical or different, are integers equal to 0, 1, 2, 3 or 4.

Derivative according to claim 1 of formula

in which :

• R ¹ corresponds to formula (V):

(V)

in which :

X represents an alkylene group forming a bridge and comprising 1,

2, 3 or 4 carbon atoms;

- Y represents a linear saturated or unsaturated C12 to Cie alkyl chain;

- Z represents:

(VI)

in which k is 1 or 2, and Y represents a saturated or unsaturated linear C12 to Cie alkyl chain,

- either a hydrocarbon group forming a bridge, comprising from 1 to 4 carbon atoms, and which may comprise one or more heteroatoms chosen from O and N, in which case R ⁴ represents a primary amine group or a group of formula (VII):

-NH-CO-Y

(VII)

in which Y represents a saturated or unsaturated linear C12 to Cie alkyl chain;

• R ² represents a hydrogen atom or a linear, branched and/or cyclic hydrocarbon group, saturated or unsaturated, comprising from 1 to 20 carbon atoms, comprising one or more heteroatoms chosen from nitrogen, oxygen, sulfur, fluorine, chlorine, bromine or iodine having at least one cationic functional group chosen from an amino, guanidino, imidazole or quaternary ammonium group; And

• R ³ represents a hydrogen atom or is identical to R ¹ , or even is identical to R ² .

3. Derivative according to one of claims 1 or 2, chosen from:

• 1, 4-didodecyl-7-betainyl-1, 4, 7-triazacyclononane;

• 1, 4-ditetradecyl-7-betainyl-1, 4, 7-triazacyclononane;

· 1, 4-dihexadecyl-7-betainyl-1, 4, 7-triazacyclononane;

• 1, -dioctadecyl-7-betainyl-1, 4, -triazacyclononane;

• 1, -dioleyl-7-betainyl-1, 4, 7-triazacyclononane; · 1, 4-didodecyl-N (7 '-carboxymethyl-1 ', 4 ', 7 ¹ -triaza- cyclononane) -L-aspartate;

• 1, 5-didodecyl-N (7 ¹ -carboxymethyl-1 ', 4 ¹ , 7 * -triaza- cyclononane) -L-glutamate;

• 1, 4-ditetradecyl-N (7'-carboxymethyl-1',4',7'-tri-azacyclononane)-L-aspartate;

• 1, 5-ditetradecyl-N (7 '-carboxymethyl-1 ', 4 ', 7 ¹ -tri- azacyclononane) -L-glutamate;

• 1, 4-dihexadecyl-N (7 ¹ -carboxymethyl-1 ', 4 ', 7 ' -tri- azacyclononane) -L-aspartate;

· 1, 5-dihexadecyl- (7 '-carboxymethyl-1 ', 4 ¹ , 7 '-tri- azacyclononane) -L-glutamate;

• 1, 4-dioctadecyl-N (7 '-carboxymethyl-1 ', ', 7' -=tri- azacyclononane) -L-aspartate; 1,5-dioctadecyl-(7*-carboxymethyl-1', ^41,7' -tri azacyclononane)-L-glutamate;

1, 4-dioleyl-N (7 '-carboxymethyl-1 ¹ , 4 ¹ , 7 '-triaza cyclononane) -L-aspartate;

1, 5-dioleyl-N (7 ¹ -carboxymethyl-1', ', 7' -triaza cyclononane) -L-glutamate;

1,-dioleyl-N ( ^11,4' -dibetainyl-7'-carboxymethyl

1', 4', 7' ÷triaza-cyclononane) -L-aspartate;

1,5-dioleyl-N ( ^11,4' -dibetainyl-7'-carboxymethyl

1',',7'-triaza-cyclononane)-L-glutamate;

1,4-dioctadecyl-7-ornithyl-1,4,7triazacyclononane;

1,4-dioleyl-7-ornithyl-1,4,7-triazacyclononane; 1,4-dioctadecyl-7-arginyl-1,4,7triazacyclononane;

1, 4-dioleyl-7-arginyl-1, 4, 7-triazacyclononane; 1, 4-dioctadecyl-7-lysyl-1, 4, 7-triazacyclononane; 1, 4-dioleyl-7-lysyl-1, 4, 7-triazacyclononane; 1, 4-dioleyl-N (1 4 '-ornithyl-1 ¹ , 4 ', 7 '-triaza cyclononane-7 '-carboxymethyl) -L-aspartate;

1,5-dioleyl-N (1','-ornithyl-1',4',7'-triaza cyclononane-7 ^1- carboxymethyl)-L-glutamate;

1,4-dioleyl-N (1',4'-arginyl-1',4', ⁷¹ -triaza cyclononane-7'-carboxymethyl)-L-aspartate; and 1,5-dioleyl-N (1','-arginyl- ¹ ', ^1,71 -triaza cyclononane-7'-carboxymethyl)-L-glutamate;

1, 4-dioleyl-N (1 ¹ , 4 '-diornithyl-7 ¹ -carboxymethyl

1',',7'-triazacyclononane)-L-aspartate;

1, 5-dioleyl- (1 ¹ , 4 '-diornithyl-7 ¹ -carboxymethyl

1 ¹ , 4', 7'-triazacyclononane)-L-glutamate;

1, 4-dioleyl-N (1 ¹ , 4 ¹ -diarginyl-7 '-carboxymethyl

1 ¹ , 4 ¹ , 7 '-triazacyclononane) -L-aspartate; • 1, 5-dioleyl-N (1 ', 4 ¹ -diarginyl-7 ¹ -carboxymethyl - 1 ', 4 ¹ , 7' -triazacyclononane) -L-glutamate;

• l-octadecyl-4-oleyl-7-betainyl-1, , 7-triazacyclononane;

• l-octadecyl-5-oleyl-N (7 '-carboxymethyl-1 ¹ , 4 ', 7 ¹ - triaza-cyclononane) -L-glutamate;

• 1-octadecyl, 4-oleyl-N (7 ¹ -carboxymethyl-1', 4', 7 ¹ - triaza-cyclononane) -L-aspartate;

• l-dodecyl-4-oleyl-7-betainyl-1, 4, 7-triazacyclononane;

• l-oleyl-4-dodecyl-7-betainyl-l, , 7-triazacyclononane.

4. Use of a derivative according to one of claims 1 to 3, as a transporter of active molecules.

5. Use according to claim 4, characterized in that the active molecule is chosen from proteins, nucleic acids or anti-tumor agents.

6. Use according to claim 5, characterized in that the active molecule is an antibody.

7. Pharmaceutical composition comprising:

- a derivative according to one of claims 1 to 3;

- a co-lipid; And

- a pharmaceutically acceptable support.

8. Nanomedicine comprising on the one hand, at least one derivative according to one of claims 1 to 3 and on the other hand, at least one active molecule.

9. Nanomedicine according to claim 8, for the treatment of autoimmune diseases.

10. Nanomedicine according to claim 9, for the treatment of cancer.

11. Product containing a nanomedicine according to one of claims 8 to 10 and at least one second active compound as a combination product for simultaneous, separate or spread administration over time, for the transport of active molecules of therapeutic interest.

12. Product according to claim 11, for the intracellular delivery of antibodies.