FR2649321A1 - Compositions based on nucleotide derivatives, methods for preparing them and their uses, in particular as pharmaceutical compositions - Google Patents

Abstract

The invention relates to compositions comprising compounds consisting of nucleotide derivatives, of therapeutic and/or clinical interest, coding, where appropriate, for a particular polypeptide, these derivatives being hydrophilic, positively or negatively charged, modified or otherwise and combined with a hydrophobic derivative possessing, where appropriate, a charge complementary to that of the abovementioned nucleotide derivatives. The invention relates more especially to pharmaceutical compositions comprising compositions as defined above, and to their uses, in particular for the treatment of viral, parasitic or neoplastic diseases.

Description

COMPOSITIONS BASED ON NUCLEOTIDE DERIVATIVES,
THEIR PREPARATION METHODS, AND THEIR
USES IN PARTICULAR AS PBARAACEUTICAL COMPOSITIONS.

 The present invention relates to compositions based on nucleotide derivatives. It also relates to the processes for preparing these compositions, as well as their use, especially in the pharmaceutical field.

 Modified nucleosides, also known as "pseudonucleosides", have been successfully used for several years against viral diseases, the most recent of which is AZT (3'-azidothymidine) used in the treatment of AIDS patients (Roland K.

ROBINS, Synthetic Antiviral Agents, C and EN (1986) 28-40). These compounds act as chain terminators in the elongation processes (reverse transcription, for example). These compounds are active only after transformation into the corresponding triphosphate derivative cells. Since this process is the limiting step in the mode of action of these drugs, it is therefore preferable to use them directly in triphosphate form.

 Unfortunately, the triphosphate form of the nucleoside badly crosses the cell membrane, which is not the case for the corresponding nucleoside.

 Other compounds of the same chemical nature but having a higher number of units per molecule, such as oligonucleotides or polynucleotides, some of which possess recognized antiviral properties.

 Indeed, the oligonucleotide derivatives, have known a considerable development these last ten years because of their ability to selectively inhibit the expression of genes (in particular genes of viruses, parasites, and oncogenes) whose products of expression are directly or indirectly involved in the development of a particular disease.

 It has also been shown that oligonucleotides of sequences complementary to certain regions of the AIDS virus genome (HIV virus) are capable of inhibiting the proliferation of this virus in vitro (S.

AGRAWAL et al., Proc. Natl. Acad. Sci., USA (1988) 85, 7079-7083).

 However, the use of these nucleotide derivatives (of natural or modified structure), in particular in cell culture or in therapeutic application, faces the problem, on the one hand, of their high sensitivity to the degradation by enzymes of the organism, such as nucleases, and, on the other hand, their low cell penetration.

These problems have been partially solved by chemical modification of the phosphodiester backbone giving rise to new classes of oligonucleotides such as phosphonate, phosphotriester, and oligonucleotides. phosphorothioates (in particular those described in French Patent No. 84.11795 filed July 25, 1984, and in S. AGRAWAL et al, cited above) and also the oligonucleotides in configuration a (C. HELENE and NT THUONG, Nucleic Acids and Molecular
Biology, vol. 2, ed. by F. ECKSTEIN and DMJ LILLEY,
Springer Verlag, 1988, pp 105-123).

 Some of these compounds thus modified actually have a relatively good resistance to nucleases. However, their cell penetration power remains very low, especially for those of these compounds which are charged, such as, for example, the oligonucleotides in α-configuration and the phosphorothioate oligonucleotides.

Finally, polynucleotides such as [poly (I): poly (C)] are known for their ability to induce interferon production (NB FINTER in "Interferons and Interferon Inducers", American Elsevier, New York, (1973) )
The object of the present invention is precisely to solve the problem of the sensitivity to nucleases and the low penetration into the cells of these nucleotide compounds, whether in natural or modified form.

 It is understood that in what precedes and what follows, the expression nucleotide derivatives, includes both mono, di or triphosphate mononucleotide derivatives, oligonucleotide derivatives and polynucleotide derivatives, whether in natural or modified form. .

 The invention relates to compositions characterized in that they comprise compounds consisting of nucleotide derivatives of therapeutic and / or clinical interest, these compounds being associated with physiologically acceptable transport vectors. These transport vectors are likely, on the one hand, to protect. said compounds of the action of the enzymes of the organism and, on the other hand, to allow the penetration of these compounds through the cell membrane of cells containing one (or more) DNA sequence, or RNA, endogenous or exogenous, this sequence being directly or indirectly involved in the process of the development of a specific pathology.

 By nucleotide derivatives of therapeutic or clinical interest forming part of the compositions of the invention, is meant more particularly any derivative capable of either being directly active against a specific disease, or to allow the implementation of a method of in vitro diagnosis of this disease.

 Among these nucleotide derivatives, there is first of all the mononucleotide derivatives consisting of a natural or modified nucleotide and comprising in their structure one, two or three phosphate groups.

 Oligonucleotide derivatives, known as "antisense" derivatives, are capable of specifically recognizing a DNA or RNA sequence (or target sequence) representative of a specific pathology and / or directly related to the process of development of this pathology. These antisense derivatives generally comprise in their structure a sequence complementary to the target sequence.

 These DNA or RNA target sequences, thus recognized, are either endogenous sequences (or else naturally present in the human or animal genome), or exogenous sequences (or else not belonging to the human or animal genome). origin, but from outside, including through bacteria, parasites, or viruses).

 In general, the derivatives of therapeutic interest included in the compositions of the invention are attached to the above-mentioned sequences so as to inhibit the development process of a specific disease caused during the expression of this sequence. In other words, they are active by "selective blocking" of the expression of a gene whose expression product is responsible for a specific disease.

By selective blocking is meant both the blocking of a sequence involved in the initiation, propagation, or termination of the replication of a gene, during the transcription of one or more genes, and / or maturation or translation of
ARN corresponding messengers.

 It can also be a protective action that makes the "blocked" sequence inaccessible to a protein, an enzyme or a chemical compound, for example.

 The derivatives of clinical interest included in the compositions of the invention are generally nucleotide probes capable of hybridizing specifically under specified conditions with DNA or RNA sequences specific for a virus, a bacterium or a parasite, following an infection of an individual, or an animal, or to a pathology resulting from a disturbance of the functioning of certain geniuses of an individual (for example tumors, pathologies related to hormonal disorders and or enzymatic).

 In general, the nucleotide derivatives (generally comprising from 1 to 50 nucleotides, and preferably from 12 to 20 nucleotides) or polynucleotides in single and double strand (generally comprising from 102 to 109 nucleotides, and preferably from 500 to 20,000 nucleotides. nucleotides) used in the context of the present invention, even if they contain certain modifications, are preferably hydrophilic and comprise charges, more particularly negative charges.

 The natural nucleotide derivatives are more advantageous to use in the context of the present invention, given their simplicity of obtaining and their low cost, that the modified nucleotide derivatives which may, for some of them, not not correctly hybridize with the target sequence.

 By way of examples, mononucleotide derivatives of particular interest in the context of the present invention include those described in the article by ROBINS et al previously cited.

 Among the oligonucleotides and polynucleotides of particular interest for use in this invention, mention will be made especially of those with antitumor activity (especially by blocking oncogenes) or with antiviral activity, or antiparasitic, in charged form, as described in French Patent No. 84.11795, and in the above-mentioned article by AGRAWAL et al.

 The above-mentioned transport vectors are preferably biocompatible and biodegradable, hydrophobic and of a polymeric or protein nature.

 Among the preferred transport vectors forming part of the compositions of the invention, mention may be made of basic proteins (such as histones) and plasma proteins, in particular lipoproteins (such as low-density lipoproteins, or LDL). , or serum albumin, monoclonal antibodies, hormones.

 By their property of recognizing highly specific epitopes on proteins expressed on the surface of well-defined cells, monoclonal antibodies, or hormones, are particularly advantageous vectors for targeted transport of these derivatives.

 In addition to the fact that lipoproteins protect the nucleotide derivatives from the action of nucleases, they also allow targeted transport of these derivatives to the target cells (especially to the cells of certain tumors) on which a large number of these receptors are expressed. lipoproteins. Given their non-immunogenic nature and their wide variety currently available in the market, lipoproteins represent very advantageous transport vectors.

 Other particularly preferred transport vectors of the invention are represented by submicroscopic polymer-based particles. For example, nanoparticles made based on isobutylcyanoacrylate polymers (as described in European Patent No. 064967 filed on April 23, 1982), or else microparticles made from copolymers of lactic acid and of glycolic acid (G. SPENLEHAUER, M. GREEN, JP BENOIT, F.

CHABOT and M. VIEILLARD, J. Control. Release, (1988) 2, 217-229) can be used for the intended application in the context of the present invention.

 Nanoparticles means any particles of the above-mentioned type and having a mean diameter of the order of about 100 to about 500 nanometers. Preferably, the nanoparticles used in the context of the present invention have a mean diameter of the order of 200 nanometers.

 These nanoparticles have the advantage of protecting the nucleotide derivatives from the effects of the nucleases of the body, and promote the penetration of these nucleotide derivatives into the cells.

 Particularly preferred compositions of the invention are those containing, as transport vector, plasma proteins as mentioned above, these proteins themselves being encapsulated in polymeric particles such as the nanoparticles defined above.

 However, given their hydrophilic nature and their charges, the preferred nucleotide derivatives of the invention can not be associated as such with the essentially hydrophobic transport vectors forming part of the compositions according to the invention.

 The present invention provides specifically hydrophilic and charged nucleotide derivatives stably associated with hydrophobic transport vectors.

 As such, the invention more particularly relates to compositions characterized in that they comprise compounds consisting of mononucleotide derivatives, oligonucleotides or polynucleotides of therapeutic and / or clinical interest, as mentioned above, these derivatives being hydrophilic, positively or negatively charged, modified or not, and associated with a hydrophobic derivative having, where appropriate, a charge complementary to that of the above-mentioned nucleotide derivatives, these compounds being themselves associated with biocompatible transport vectors, in particular polymeric and / or proteinic nature as defined above.

 The above-mentioned hydrophobic derivative is advantageously biocompatible and biodegradable.

 Among the hydrophobic, non-charged derivatives that can be used in the context of the present invention, mention will be made of the derivatives of porphyrin, daunomycin, lipids, etc., which hydrophobic derivatives are linked to the nucleotide derivative via a covalent bond.

 Particularly advantageous hydrophobic derivatives, in particular. in the case where the nucleotide derivatives used are charged, are in ionic form (anionic or cationic) and have charges complementary to those of the derivatives to be associated with the transport vectors.

These hydrophobic derivatives are capable of binding on the one hand to nucleotide derivatives, by electrostatic interaction with the charge of these latter derivatives, and on the other hand with the hydrophobic regions of the vector by their hydrophobic portion.

dans laquelle - X représente un halogène, notamment un atome de chlore ou de brome, - Y représente un atome d'azote ou de phosphore.By way of example of preferred hydrophobic ions of the invention, mention will in particular be made of the cations derived from the salts of general formula

in which - X represents a halogen, especially a chlorine or bromine atom, - Y represents a nitrogen or phosphorus atom.

the groups R 1, R 2, R 3 and R 4, which may be identical or different, represent a hydrogen atom, or a hydroxyl function, or a halogen or a linear or branched alkyl or alkoxy group having from 1 to 25 carbon atoms; optionally substituted by one or more hydroxyl functions, or an aryl group, especially a phenyl or polyaromatic group, which may contain one or more heteroatoms such as N, S, P
Se, etc ..., - at least one of these groups R1, Rz, R3, or Rq represents an alkyl or aryl group as defined above.

 It goes without saying that the ammonium salts (in which Y represents a nitrogen atom) or phosphonium (in which Y represents a phosphorus atom) mentioned above are particularly suitable for the preparation of compositions of the invention comprising negatively charged nucleotide derivatives.

In the case where the nucleotide derivatives are positively charged, one will use as hydrophobic derivative an anion, in particular that of the sodium tetraphenylboride of formula

 Other particularly preferred hydrophobic derivatives of the invention are fatty-chain amines having a positive charge, at physiological pH, and the hydrocarbon-based chains of which contain in particular 5 to 25 carbon atoms, for example stearylamine, sphingosine, or by lipids or phospholipids, and whose hydrocarbon chains include in particular from 10 to 25 carbon atoms also having a positive charge in their structure.

 The combination of the nucleotide derivative with a charged hydrophobic derivative as described above is advantageously made, by simple contacting of the hydrophobic derivative, having a charge complementary to that of the nucleotide derivative, with said nucleotide derivative under conditions allowing the formation of an electrostatic type bond between these two derivatives.

 This latter solution represents a preferred variant of the process for preparing the compositions of the invention. Indeed, the establishment of a simple electrostatic bond between the hydrophobic derivative and the nucleotide derivative in no way modifies the structural and functional properties of the latter, unlike a covalent bond which risks modifying the properties mentioned above. mentioned and therefore alter the therapeutic and / or diagnostic effects of said nucleoside or nucleotide derivatives.

In the context of this preferred variant of the process of the invention, the step of contacting the nucleotide derivative with the hydrophobic derivative in ionic form is advantageously carried out so that the ratio
charge of the hydrophobic derivative r =
charge of the nucleotide derivative, between 1 and 200, preferably between 10 and 50.

 The subject of the invention is also a process for the preparation of these compositions comprising mainly the following steps - where appropriate, the bringing into contact of the nucleotide derivative with a hydrophobic derivative, preferably charged, when said nucleotide derivative is hydrophilic and charged, in conditions, as described above, permitting the formation of a bond between them, it being understood that the expression "binding" is understood as the result of mutual affinity, in particular chemical (for example resulting from a coupling), or physical (complexation for example) or electrostatic, - the incorporation of the compound consisting of the nucleotide derivative linked, where appropriate, to the above-mentioned hydrophobic derivative, in the transport vector, by mechanical mutual interpenetration, in particular by encapsulation or by adsorption of one into the other.

 In the case of the use of a transport vector of a polymeric nature, the encapsulation step of the compound consisting of the nucleotide derivative linked, where appropriate, to the hydrophobic derivative, in this vector is carried out either by bringing into the presence of said compound with the monomer units of the vector followed by a step of polymerization of said vector then including said compound, either during the course of the polymerization, that is to say after the start of the polymerization but before the completion of the latter, or by bringing said compound into contact with the vector in polymerized form, provided that the conditions of this setting can be adjusted so as to allow a sufficiently strong physical retention (or adsorption) of said compound on the polymer, so that the compound adsorbed or retained on the polymer can reach at the same time as the latter the target cells.

 Optimum conditions in which the encapsulation of said compound in submicroscopic particles, in particular in alkylcyanoacrylate nanoparticles such as isobutylcyanoacrylate nanoparticles, may be carried out, are more particularly described in the aforementioned European patent 064967, as well as in the detailed description that follows.

 Thus, the subject of the invention is more particularly a process for the preparation of a composition as described above, which comprises the addition, with stirring, of the compound bound, where appropriate, to a hydrophobic derivative, to a solution comprising a polymer of the above-mentioned type, either in monomeric form, or during polymerization, or in polymerized form.

 It may be further specified that in the context of the use of the nanoparticles mentioned above, the step of encapsulation of said compounds will advantageously be in solution at a physiological pH close to 7, and at NaCl concentrations (0.15 M) close to physiological saline concentrations.

 The adsorption of said compounds to plasma proteins, in particular lipoproteins, is advantageously under the conditions similar to those set out above.

 The invention also relates to a preparation process comprising a step of adsorbing said compounds (consisting of nucleotide derivatives associated, where appropriate, with a hydrophobic derivative), to plasma proteins, in particular lipoproteins, followed by a step of encapsulation of the previously obtained complex in polymeric particles, especially in nanoparticles as defined above.

 The invention also relates to pharmaceutical compositions comprising at least one of the above-mentioned compositions in association with a physiologically acceptable vehicle.

Such pharmaceutical compositions are more particularly intended for the treatment of diseases of viral (especially AIDS), bacterial, parasitic, or tumoral origin or any other pathology related to a modification of the genome of an individual.
The dosages and routes of administration of these compositions may vary depending on the type of infection to be treated.

 In the most general case, the pharmaceutical compositions of the invention are in the form of injectable preparations in combination with a pharmaceutically acceptable liquid, at physiological pH.

 The subject of the invention is also a method of cellular transfection comprising contacting a composition according to the invention comprising a specific nucleotide derivative, in particular one or more determined genes, with cells to allow the internalization of the nucleotide derivative known above. -mentioned in said cells.

 The invention more particularly relates to a transfection method as defined above, wherein said determined nucleotide derivative comprises a nucleotide sequence. coding for a specific polypeptide, and, where appropriate, regulatory elements allowing the expression of this nucleic acid fragment in the host cell, in particular a promoter recognized by the polymerases of said host cell. Obtaining the determined polypeptide is done by culturing the transformed host cells in an appropriate culture medium, followed by recovering the determined polypeptide from these cells or the culture medium.

 Thus, the subject of the invention is compositions as described above in which the nucleotide derivative associated with the transport vector, and, where appropriate, with a hydrophobic derivative, codes for a specific polypeptide.

 The subject of the invention is more particularly compositions of the above-mentioned type in which the determined nucleotide derivative is inserted into the nucleotide sequence of a gene vector, in particular a plasmid, this plasmid itself being associated with a derivative. hydrophobic mentioned above, the assembly consisting of this plasmid associated with the hydrophobic derivative, itself being associated with a transport vector as described above.

 This nucleotide derivative included in this plasmid may also have a nucleotide structure such as to block the synthesis of a specific protein. In this case, such a composition is particularly advantageous, in combination with a physiologically acceptable vehicle, for the treatment of pathologies related to the synthesis of one (or more) determined undesirable protein.

 Other features of the invention will become apparent from the following description of an example of obtaining compositions of the invention. It goes without saying that these examples are not limiting in nature.

I - OLIGONUCLEOTIDES-NANOPARTICLES ASSOCIATION
Standard experience
In 1 ml of polymerization medium containing 1% Dextran 70 and the necessary amount of HCl so that the pH of the medium is equal to 3, 10 l of isobutyl cyanoacrylate monomer (IBCA) is added with vigorous magnetic stirring to disperse the monomer. After 10 to 15 minutes in which the polymerization results in an opalescence of the medium, the ethanolic solution (80 μl to 200 μl) containing the complex between the mono or oligonucleotide and the hydrophobic ion is added, studied in proportions defined by r, r being equal to the ratio of positive charges to negative charges of the oligonucleotide. The mixture is left in contact with the nanoparticles being formed for 45 minutes with stirring. The medium is then neutralized with 7 μl of 1 M phosphate buffer adjusted to pH 7, then 30 μl of 5 M NaCl are added to adjust the ionic concentration of the medium to 0.15 M
NaC1. The mixture is allowed to run for 30 minutes to equilibrate and the solution is centrifuged at 35000 g at 10 ° C.

Since the oligonucleotide is labeled at one end by phosphorus 32, the measurement of the radioactivity of the supernatant S and of the pellet C makes it possible to determine the degree of encapsulation r = 100 (C / C + S).

The hydrophobic ions used in the following tests are shown in Tables 1 to 3 below
TABLE 1
Code Formula Compound: ST: CH3- (CH2) 17-NH + 3: Stearylamine (1-amino-octadecane)
SP CH3- (CH2) 14-CHOH-CH-CH2OH DL-Dihydrosine
NH3 (DL-1,3dihydroxy2-amino-octadecane Table 2

<SEP>#<SEP> X
<tb> Code <SEP> R1 <SEP> R2, R3, R4 <SEP> x <SEP> COMPOUND
<tb> A1 <SEP> CH3 <SEP> CH3 <SEP> Cl <SEP> Tetramethylammonium, <SEP> Chloride
<tb> A2 <SEP> n-C4H9 <SEP> n-C4H9 <SEP> Cl <SEP> Tetrabutylammonium, <SEP> Chloride
<tb> A3 <SEP> n-C6H13 <SEP> n-C6H13 <SEP> Cl <SEP> Tetrahexylammomum, <SEP> Chloride
<tb> A4 <SEP> n-C8H17 <SEP> n-C8H17 <SEP> Cl <SEP> Tetraoctylammonium, <SEP> Chloride
<tb> A5 <SEP> n-C16H33 <SEP> CH3 <SEP> Br <SEP> Hexadecyltrimethylammonium,
<tb><SEP> bromide <SEP> (CTAB)
<tb> Table 1

<SEP>#<SEP> X
<tb> Code <SEP> R1 <SEP> R2, R3, R4 <SEP> x <SEP> COMPOUND
<tb> P1 <SEP> CH3 <SEP> CH3 <SEP> Cl <SEP> Tetramethylphosphonium, <SEP> Chloride
<tb> P2 <SEP> n-C4H9 <SEP> n-C4H9 <SEP> Cl <SEP> Tetrabutylphosphonium, <SEP> Chloride
<tb> P3 <SEP> C6H5 <SEP> C6H5 <SEP> Cl <SEP> Tetraphenylphosphonium, <SEP> chloure
<tb> P4 <SEP> n-C16H33 <SEP> n-C4H9 <SEP> Br <SEP> Hexadecyltributylphosphonium,
<tb><SEP> bromide <SEP> (CTPB)
<Tb>
A) Encapsulation of adenosine triphosphate
The encapsulation tests of adenosine 5'triphosphate (ATP) or didesoxyadenosine 5'triphosphate (dd-ATP) in the presence of long-chain amines 9 such as stearylamine or sphingosine has made it possible to achieve encapsulation levels. important (Table 4). In these experiments, the label used is the corresponding 32-phosphorus-labeled radioactive derivative.

TABLE 4
N experiment Cation XTP (a) r: 1:: ATP: 200: 55:: Stearylamine:: 2:: dd-ATP: 200: 46: 3:: ATP: 200: 73: Sphingosine: 4:: dd-ATP : 200: 77 (a): The XTP concentration is kept constant
and equal to 1.50 x 10-5 M phosphate and that of
cation at 3 x 10-3 M from which r = 200.

B) Encapsulation of oligonucleotides
1. Octathymidylate:
The concentration of p (dT) is kept constant and equal to 1.5 × 10 5 M phosphate or 1.9 μM oligonucleotide. In the following experiments, the oligonucleotide is 32 P labeled at the 5 'end and purified by polyacrylamide gel electrophoresis.

The variations in the encapsulation rates of the cation labeled P3 (cf Table 3) with carbon 14C and p (dT) 8 (labeled with 32p) (double labeling method) as a function of the concentration of P3 are shown in FIG. In this figure, the values of r represent those obtained without addition of NaCl at the end of polymerization. The values of r presented in the other tables and figures are obtained in the presence of 0.15 M final NaCl. In Table 5, the results show that in many cases, the oligonucleotide can be encapsulated at more than 90%, while avoiding the phenomenon of flocculation of the nanoparticles.

TABLE 5
N of experience: Cation: r: r (8)
5: 200: 98f
Tetraoctyl N + (A4)
6 50 31.3
7:: 200: 90
CTAB (A5)
8 50 66
9:: 200: 68.4
Tetraphenyl P + (P3)
10 50 41.6
11:: 200:
CTPB (P4)
12 50 31.6
13:: 200: 95f
Stearylamine (ST)
14 50 51.5
15:: 200: 96.8
16: Sphingosine (SP): 50: 88
17:: 20: 41,6 f: Observation of aggregation of nanoparticles or
flocculation at high cation ratios
hydrophobic.

2. Hexadecathymidylates P (dT) rZ
In these experiments, the concentration of oligonucleotide is 1.84 M or 2.94 × 10 5 phosphate. The results presented in Table 6 show that cations containing a long hydrocarbon chain make it possible to reach high encapsulation rates (> 95%) for relatively low ratios (r = 20). In addition, the use of low concentrations of cation makes it possible to produce stable suspensions of nanoparticles except in the case of DOTMA. The evolution of the encapsulation rate (r) as a function of the ratio r (number of cations per phosphate) is presented in FIG. 2 for the 3 cations A5, ST and SP.

TABLE 6
N of experience Cation r @ (%)
18 Tetraoctyl N + (14) 20 61.4
19 CTAB (15) 20 98
20 CTPB (P4) 20 96.7
21 Stearylamine (ST) 20 96
22 Sphingosine (SP) 20 97.5
DOTMAa 7 69.5f a: N- [1 (2,3-dioleyloxy) propyl] -N, N, N-trimethylam
monium, chloride.

f: flocculation.

3. Encapsulation in the presence of sphingosine (SP) of a few oligonucleotides of known sequence including an oligophosphorothioate
The results are shown in the following Table 7.

 The concentration of oligonucleotide used is 0.102 M in oligonucleotide.

TABLE 7
NOT . Experience: Cation: r: r (%)
24: 5'-ATAGTACAGAAA-3 ': 50: 49
(PO)
25: 5'-ATAGTACAGAAA-3 ': 50: 81.2
(PS)
26 p (dT) 16 50 30.9
27: S'TCCTGATAAAGGA-: 50: 36.2
: GGAGATGAAG
: AAAAAATGA-3 'a: oligophosphorothioate in which a non-bridging oxygen atom is replaced by a sulfur atom.

 These experiments show that at identical length and sequence, an oligophosphorothioate encapsulates better than the corresponding phosphodiester compound. By comparing experiments 26 and 22, it can be seen that the encapsulation efficiency is directly related to the concentration of the cation-oligonucleotide complex present in the polymerization medium.

 4) Direct encapsulation of some modified oligonucleotides.

 In the following experiments, the results of direct encapsulation (absence of hydrophobic cations) of some oligonucleotides at the end of which are covalently attached various chemical groups (see Table 8): hydrocarbon chain, intercalating (derivatives of the acridine), drug (daunomycin), photosensitizer (porphyrin). The structure of these compounds is shown in FIG. 3. The presence of these groups can confer on the hybrid molecule an increased hydrophobic character which is reflected in their level of encapsulation in the nanoparticles as well as in their association with lipoproteins. (to see further).

TABLE 8
N Oligonucleotide Experiment (ON) [ON] # (%)
28 μ (dT) 8 0.5 M 3: 29: T8- (CH 2) 5-Acridine: 0.5 M: 5: 30: T 7 (CH 2) 5-Porphyrin: 0.5M: 98 31 5'-TTTTCCTCCTCT -dauno
mycine 2 M 79: 32: 5'-AGCCACACC- (CH2) 11-
CH3 12 M 3
In experiments Nos. 29, 30 and 31, the encapsulation is followed by fluorimetric measurements.

 It has been verified that the emission properties of the fluorophores are not affected by the presence of the polymer.

c) Encapsulation of polynucleotides
In the following examples, it clearly appears that the poly (isobutylcyanoacrylate) nanoparticles can encapsulate single-stranded polynucleotides whether they consist of ribo- (polyU) structure units or deoxyribonucleotide units such as poly (dT) (see Talbeau 9). ). The example of the double-stranded structures is illustrated by calf thymus DNA (not sonicated) (experiment nos. 36 and 37).

TABLE 9
NX experiment: Cation: PolyN: r: r (%) 33: Sphingosine: poly (dT): 3: 28
(sp): 34: Tetraoctyl N +: polyU: 10: 52 (A4) 35: CTAB (A5): polyU: 20: 54
36: CTAB (A5); DNA: 50: 76: 37: Sphingosine: DNA; 50: .96: (SP)
The concentration of the polynucleotide is 4 x 10-5 M in phosphate. These experiments show that it is possible to use these polymer vectors to encapsulate messenger RNAs or DNA of higher organisms whose molecule may contain several billion base pairs.

 II Association oligonucleotides-lipoproteins.

 Lipoproteins represented in this example by the low density fraction, LDL fraction, can bind oligonucleotides when they carry a hydrophobic group such as porphyrin (FIG. 4).

The results show that an LDL molecule for example can bind up to 17 molecules of a porphyrin-coupled oligoheptathymidylate (T7-POR).

In a control experiment under the same conditions, an uncoupled oligonucleotide (pdT16) is not encapsulated.

 The use of vectors such as LDL could target oligonucleotides to certain types of tumor cells where LDL receptors are expressed in greater numbers than untransformed parent cells.

 The legends of the figures mentioned above are as follows - FIG. 1: encapsulation of P3 (o) and p (dT) 8 (o) as a function of the concentration of P3. The concentration of oligonucleotides is kept constant and equal to 1.6 × 10 6 M phosphate and that of nanoparticles (NP) 8 mg / ml. The pH of the polymerization medium is adjusted to 7 after 1 hour of polymerization by addition of 7 l of sodium phosphate buffer (1 M). In this experiment, there is no addition of NaCl at the end of polymerization.

stearylamine (o) ou CTAB

par des nanoparticules d'IBCA (10 mg/ml). Le pH du milieu de polyméristion est ajusté à 7 puis équilibré avec NaCl (0,15 M final) en fin de polymérisation.FIG. 2: encapsulation of p (dT) 16 (2.94 × 10 5 M phosphate) in the presence of sphingosine

stearylamine (o) or CTAB

by nanoparticles of IBCA (10 mg / ml). The pH of the polymerization medium is adjusted to 7 and then equilibrated with NaCl (final 0.15 M) at the end of the polymerization.

FIG. 4: Oligonucleotide binding curve
T7-POR on LDL (human plasma) determined by measuring the fluorescence of porphyrin at 626 nm. The concentration of oligonucleotide is 3.5 μM. The stoichiometry of the complex is estimated at 17 fixed oligonucleotide molecules per LDL molecule.

Claims (13)

 1. Composition characterized in that it comprises compounds consisting of nucleotide derivatives of therapeutic and / or clinical interest, coding, where appropriate, for a specific polypeptide, these derivatives being hydrophilic, positively or negatively charged, modified or unmodified. , and associated with a hydrophobic derivative having, if necessary, a charge complementary to that of the above-mentioned nucleotide derivatives.  2. Composition according to claim 1, characterized in that the nucleotide derivatives consist of a nucleotide sequence inserted into the sequence of a vector, in particular a plasmid.  3. Composition according to claim 1 or claim 2, characterized in that the hydrophobic derivative is not loaded, and is in particular a derivative of the porphyrin type, or daunomycin.  4. Composition according to claim 1 or claim 2, characterized in that the hydrophobic derivative is charged and is in cationic or anionic form.  5. Composition according to claim 4, characterized in that the ratio  charge of the hydrophobic derivative r =  charge of the nucleotide drift, is between 1 and 200, preferably between 10 and 50.  6. Composition according to claim 4, characterized in that the hydrophobic derivative corresponds to the cation derived from the compound of general formula

 in which - X represents a halogen, especially a chlorine or bromine atom, - Y represents a nitrogen or phosphorus atom, - the groups Rr, R2, R31 and R4, identical or different, represent a hydrogen atom or a hydroxyl function, or a halogen, or a linear or branched alkyl or alkoxy group having from 1 to 25 carbon atoms, or an aryl group, in particular a phenyl group, or a polyaromatic group that may contain one or more heteroatoms such as N , S, P, Se - at least one of these groups R1, R21 R31 or R4 represents an alkyl or aryl group as defined above.  7. Composition according to Claim 4, characterized in that the hydrophobic derivative corresponds to the anion derived from sodium tetraphenylboride of formula

 8. Composition according to any one of claims 1 to 7, characterized in that the transport vectors consist of nanoparticles based on polymer, in particular an alkylcyanoacrylate polymer, or an acid-based polymer. lactic and glycolic acid.  9. Composition according to any one of claims 1 to 8, characterized in that the transport vectors consist of plasma proteins, including low density lipoproteins (LDL).  10. Composition according to any one of claims 1 to 9, characterized in that the transport vectors consist of plasma proteins, including low density lipoproteins (LDL), themselves encapsulated in nanoparticles as defined in the claim 8.  11. Pharmaceutical composition, characterized in that it comprises a composition according to any one of claims i to 10, in association with a physiologically acceptable vehicle.  12. Pharmaceutical composition according to claim 11, characterized in that it is more particularly suitable for the treatment of diseases of viral, bacterial, parasitic or tumoral origin.  13. Cell transfection method, characterized in that it comprises contacting a composition according to any one of claims 1 to 10 with host cells, under conditions permitting the internalization of the nucleotide derivative included in said composition within said host cells.