WO1996034008A1

WO1996034008A1 - Novel antisense nucleic acids directed against ras oncogenes, their preparation and use

Info

Publication number: WO1996034008A1
Application number: PCT/FR1996/000652
Authority: WO
Inventors: Claude Helene; Piet Herdewijn; Ester Saison-Behmoaras; Arthur Van Aerschot; Thanh Thuong Nguyen
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm); Centre National De La Recherche Scientifique (Cnrs); Museum National D'histoire Naturelle
Priority date: 1995-04-28
Filing date: 1996-04-29
Publication date: 1996-10-31
Also published as: FR2733500B1; FR2733500A1

Abstract

The present invention concerns single-strand antisense nucleic acids complementing a region of an mRNA of a human RAS oncogene with localized mutation, comprising between 8 and 20 nucleotides and a group of the formula -(CHR)n-OH in position 3' and/or 5', in which n is an integer between 1 and 20 inclusive and R is a hydrogen atom or an OH group.

Description

The present invention relates to antisense nucleic acids directed against the messenger RNAs of ras oncongenes, their preparation and their use, in particular for the treatment of tumor diseases. It also relates to pharmaceutical compositions comprising said antisense nucleic acids, optionally adsorbed, included or encapsulated in nanoparticles. It relates more particularly to modified antisense nucleic acids capable of specifically inhibiting the expression of mutated ras oncogenes. Different genes, called oncogenes and suppressor genes, are involved in the control of cell division. Among these, the ras genes, and their products generally designated p21 proteins, play a key role in controlling cell proliferation in all the eukaryotic organisms where they have been sought. In particular, it has been shown that certain specific modifications of these proteins cause them to lose their normal function and lead them to become oncogenic. Thus, a large number of human tumors have been associated with the presence of modified ras genes, and in particular of ras genes possessing point mutations, most often at positions 12, 13 and 61. Similarly, an overexpression of the p21 proteins can lead to a disruption of cell proliferation. Understanding the exact role of these p21 proteins in cells, and developing methods to inhibit their activity in tumor cells therefore constitute a major challenge for the therapeutic approach to cancer.

One of the approaches proposed in the prior art for inhibiting the activity of ras oncogenes lies in the use of antisense oligonucleotides. The regulation of the expression of target genes by means of antisense oligonucleotides indeed constitutes a therapeutic approach in increasing development. This approach is based on the capacity of oligonucleotides to hybridize specifically to regions complementary to a nucleic acid, and thus to specifically inhibit the expression of specific genes. This inhibition can occur either at the translational level (antisense oligonucleotide) or at the transcriptional level (anti-gene oligonucleotide). More specifically, the antisense nucleic acids are nucleic sequences capable of hybridizing selectively with cell-target messenger RNAs, to inhibit their translation into protein. These oligonucleotides form, with the target mRNA, locally, double-stranded regions, by interaction of the classic Watson-Crick type. They may for example be synthetic oligonucleotides, of small size, complementary to cellular mRNAs, and which are introduced into the target cells. Such oligonucleotides have for example been described in patent No. EP 92 574. They may also be antisense genes whose expression in the target cell generates RNAs complementary to cellular mRNAs. Such genes have for example been described in patent No. EP 140,308. However, the in vivo use of antisense nucleic acids directed against the ras genes encounters a certain number of difficulties which limit their use to date. therapeutic exploitation. First of all, nucleic acids are very sensitive to degradation by enzymes in the body, such as nucleases, which implies the use of large doses. In addition, they have low penetration in certain cell types and often inadequate intracellular distribution, which makes them without therapeutic effect. Finally, it is important to be able to have sufficiently selective and stable sequences to obtain a specific effect without altering other cellular functions.

In an attempt to solve some of these problems, it has been proposed to chemically modify the phosphodiester backbone of nucleic acids, to give rise to new classes of artificial oligonucleotides. Among these, mention may be made of oligonucleotides phosphonates, phosphotriesters, phosphoramidates and phosphorothioates which are described, for example, in patent application WO94 / 08003, oligonucleotides coupled to different agents such as cholesterol, a peptide, a cationic polymer , etc. However, if some of these modified compounds have good resistance to nucleases, their power of penetration and distribution in the various cell compartments remains very low. In addition, their biological activity is generally not increased and they have certain side effects linked to the presence of unnatural patterns in their structure. The present invention offers a particularly advantageous solution to these problems. The present invention indeed provides new nucleic acids antisense directed against ras, presenting a particular chemical modification. In a particularly advantageous manner, the antisenses according to the invention exhibit not only a high resistance to nucleases, but also a good cellular penetration followed by an appropriate distribution in the cells, a very high selectivity with respect to their target, and form particularly stable complexes with their targets.

A first object of the present invention resides in a single-stranded antisense nucleic acid complementary to a region of an mRNA of a human ras oncogene carrying a point mutation, characterized in that it comprises 8 to 20 nucleotides and a group of formula - (CHR) _n -OH in 3 ′ and or 5 ′, in which n represents an integer between 1 and 20 inclusive, and R represents a hydrogen atom or an OH group, R possibly varying from one group (CHR) to another.

Surprisingly, the applicant has now shown that it is possible to obtain in vitro selective antiproliferative effects using short antisense nucleic acids chemically modified by the addition of a - (CHR) _n -OH group at 3 ′ and / or in 5 '. The results presented in the examples further show that the antisense thus obtained have an increased resistance to nucleases and a higher biological activity. In addition, they do not induce any apparent cytotoxicity and are devoid of significant side effects. The results obtained confirm that these properties are completely unexpected insofar as the antisense of the invention are active at concentrations up to 500 times lower than those necessary for other types of antisense (cholesterol, polymer, etc. ) including natural oligophosphodiesters.

As indicated above, the group of formula - (CHR) _n -OH can be coupled 3 ', 5' or 3 'and 5' of the antisense nucleic acid. In general, the antisense and the group - (CHR) _n -OH are synthesized separately, then assembled by conventional chemical reaction. A method for coupling the - (CHR) _n -OH group in each of these positions is given in the examples. Preferably, in the formula - (CHR) _n -OH, n is an integer between 3 and 12 inclusive. Particularly interesting results have been obtained with antisense modified by a group dodecanediol (n = 12), propanediol (n = 3), dimethyl-1,3-propanediol (n = 5), or glycerol (n = 3).

The nucleic acids used in the context of the present invention have a length advantageously between 8 and 20 nucleotides. The Applicant has indeed shown that it was more advantageous to obtain a selective effect combined with good stability, to use relatively short oligonucleotides. As illustrated in the examples, the selectivity of the anti-ras anti-sense nucleic acid is particularly important when the oligonucleotide has a length of less than 14 bases. It is therefore particularly advantageous to use nucleic acids of size between 8 and 13 bases. Even more preferably, the nucleic acids used in the context of the present invention have a length of between 10 and 13 nucleotides. Particularly advantageous results have been obtained with nucleic acids of 12 nucleotides. By way of example, there may be mentioned the antisense nucleic acids of sequence SEQ ID No. 1 (AS2); SEQ ID No. 10 (AS4); SEQ ID No. 14 or SEQ ID No. 15 which have very remarkable properties. The following modified antisense are very particularly preferred within the meaning of the present invention: AS2-3'ml2OH;5'ml2OH-AS2;AS2-3'm3OH;5'm3OH-AS2;5'ml2OH-AS2-3'ml2OH,AS4-3'ml2OH;5'ml2OH-AS4;AS4-3'm3OH;5'm3OH-AS4 and 5'ml2OH-AS4-3'ml2OH, AS2-3'Glycerol; AS4-3 'Glycerol. In a particularly preferred embodiment of the invention, the nucleic acids used are chosen so that the point mutation targeted on the ras oncogene is located at the center of the complementary region. The Applicant has indeed shown that it was particularly advantageous for the antisense nucleic acid used to be centered around the targeted mutation. As illustrated in the examples, when the nucleic acid is centered on the mutation, a selective inhibition of 100% is obtained, whereas this is only 50% when the mutation is shifted only by 2 bases. These particularly surprising results make it possible, for a given target region, to prepare much more effective antisense nucleic acids. Furthermore, to further improve the therapeutic efficacy of the antisense of the invention, other chemical modifications can be combined with the presence of group (CHR) _n -OH. In particular, the combination with an intercalating agent, such as acridine, makes it possible to further increase the potential of the antisense (see examples). The intercalating agent can be introduced within the antisense or at one of the ends, or at both. Particularly advantageous results have been obtained with an antisense modified by a 5 'and 3' acridine group (Acr-ASml2OH). The synthesis of this oligonucleotide was carried out by first coupling the dodecanediol group in 3 'according to the techniques illustrated in the examples, then the acridine group in 5' according to the techniques described by Hélène in applications EP 117777 and EP 169787 by example, or according to any other protocol known to those skilled in the art. It is also possible to couple the acridine group first and then the dodecanediol group.

In a particular embodiment of the invention, the antisense nucleic acids can be adsorbed, included or encapsulated in nanoparticles. The nanoparticles which can be used in the context of the present invention can be any small particle, generally less than 500 nm, consisting of biocompatible polymer (s), capable of transporting or vectorizing an active principle in cells or in the blood circulation. Preferably, the nanoparticles according to the invention consist of polymers comprising a majority of degradable units such as polylactic acid, optionally copolymerized with polyethylene glycol, or alkyl cyanoacrylates. Other polymers which can be used in the production of nanoparticles have been described in the prior art (see for example EP 275 796 and EP 520 889). As an example, nanoparticles consisting of polymers of alkyl cyanoacrylates (as described in European patent EP 0 007 895 and French patent 8 08 172) or of polymers of lactic acid or of copolymers of lactic acid and glycolic acid (as described by G. Spenlehauer et al., J. Control. Release, (1988), 7, 217-229; EP 520888; EP 520 889).

The nanoparticles according to the invention generally have an average diameter of the order of 50 to 500 nanometers. Preferably, the nanoparticles used in the context of the present invention have an average diameter of the order of 150 nanometers. 96/34008

Of particular interest are the nanoparticles obtained by polymerization of methyl, ethyl, isobutyl or isohexyl cyanoacrylates or those obtained by copolymerization of lactic acid and glycolic acid.

Furthermore, prior to their encapsulation, the nucleic acids of the invention are generally associated with cationic hydrophobic compounds. The use of such compounds in fact makes it possible to facilitate the association of the nucleic acids with the nanoparticles. More particularly, the cationic hydrophobic compounds used in the compositions according to the invention make it possible to form pairs of ions between the phosphorus derivatives negatively charged of the nucleic chain and the hydrophobic cations and thus to increase the lipophilic character and the adsorption nucleic acids to nanoparticles. In general, the cationic hydrophobic compounds which can be used in the preparation of the compositions according to the invention are chosen from tetraphenylphosphonium halides, preferably chloride; quaternary ammonium salts, chosen more particularly from trimethylalkylammonium halides such as chlorides or bromides of dodecyltrimethylammonium, tetradecyltrimethylammonium, hexadecylrimethylammonium or cetyltrimethylammonium; fatty amines, such as D, L-dihydrosphingosine (D, L-dihydroxy-1,3 amino-2 octadecane), or oligopeptides, such as polylysine and oligopeptides of formula (LKKL) n, (LKLK) n or (LRRL) n. Particularly advantageously, in the context of the invention, a hexadecylrimethylammonium salt or an oligopeptide is used

The nanoparticulate compositions according to the invention can be obtained by any method making it possible to adsorb, to include or to encapsulate a nucleic acid, associated or not with a cationic hydrophobic compound, in a nanoparticle.

Generally, the nanoparticulate compositions according to the invention can be obtained

by bringing the nucleic acid and possibly the cationic hydrophobic compound into contact with the nanoparticles under conditions which allow strong retention (or absorption) of the nucleic acid on the nanoparticles and easy reaching of the target cells, either by bringing the nucleic acid and optionally the cationic hydrophobic compound into contact with the monomer units, followed by polymerization,

- Or by bringing the nucleic acid and optionally the cationic hydrophobic compound with the polymer during the polymerization, that is to say after the start of the polymerization but before the completion thereof.

Preferably, the compositions according to the invention are obtained by adsorption, inclusion or encapsulation on the nanoparticles already formed.

The alkyl polycyanoacrylate nanoparticles can be prepared under the conditions described in European patent EP 0007 895 and the French patent 81 08172 and those of polylactic-polyglycolic acid under the conditions described by G. Spenlehauer et al., J. Control . Release, (1988), 7, 217-229.

The adsorption of the nucleic acid complexed or not by the cationic hydrophobic compounds on the nanoparticles is generally carried out by stirring a suspension of nanoparticles with the nucleic acid complexed by the cationic hydrophobic compound under determined conditions.

The nanoparticles thus transformed are generally separated by filtration or centrifugation and are used to prepare therapeutically useful pharmaceutical compositions.

Generally, adsorption is carried out in an aqueous medium, the pH of which is buffered to a value close to 7.

As a buffer, the TRIS-HC1 buffer can be used.

Furthermore, to stabilize the suspension, it may be useful to operate in the presence of a stabilizing agent such as a synthetic non-ionic polyoxyethylene-polyoxypropylene copolymer such as the poloxamer 188 or a polyoxyethylene. This stabilizing agent makes it possible in particular to reduce the phenomena of adhesion between particles leading to the formation of aggregates.

The adsorption is generally carried out at a temperature in the region of 20 ° C. and the reaction is terminated after a few hours of stirring.

It is particularly advantageous to use, at the start of the addition operation, a concentration of nanoparticles of between 0.5 and 50 mg / cm3 and preferably close to 1 mg / cm3. The concentration of nucleic acids is generally between 0.01 and 500 μM. The concentration of nucleic acid chosen essentially depends on the concentration of nanoparticles and the desired final concentration.

Furthermore, to increase the adsorption of nucleic acids on the nanoparticles, it may be advantageous to operate in the presence of an excess of cationic hydrophobic compound.

In general, the efficiency of nucleic acid adsorption to nanoparticles depends essentially on the length of the nucleotide chain and the density of charges on the surface of the particles. Another subject of the invention relates to pharmaceutical compositions comprising an antisense nucleic acid as defined above, in combination with one or more pharmaceutically acceptable diluents or adjuvants. The antisense can be encapsulated or not, it being understood that even in free form, it has quite remarkable antiproliferative properties in vivo. The compositions according to the invention can be used in vitro, ex vivo or in vivo. In vitro, they can make it possible to transfer antisense nucleic acids to cell lines, for example in order to study the regulation of ras proteins and the ras-dependent signaling pathway. Ex vivo, they can be used for the therapeutic transfer of anti-ras anti-sense nucleic acids into a cell originating from an organism, with a view to conferring on said cell resistance to ras oncogenes, before its re-administration to an organism. In vivo, they can be used for direct administration of anti-ras anti-sense nucleic acids. In particular, intra-tumor administration is entirely advantageous since it allows the active composition to be delivered efficiently and locally. Preferably, the pharmaceutical compositions of the invention therefore contain a pharmaceutically acceptable vehicle for an injectable formulation, in particular for an intratumoral injection. They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes. The compositions according to the invention being capable of modulating the activity of ras oncogenic proteins, they make it possible to inhibit the development process and can thus be used for the treatment of various cancers. Many cancers have in fact been associated with the presence of ras oncogenic proteins. Among the cancers most often containing mutated ras genes, mention may be made in particular of pancreatic adenocarcinomas, 90% of which have a Ki-ras oncogene mutated on the twelfth codon (Almoguera et al., Cell 5_3 (1988) 549), colon adenocarcinoma and thyroid cancer (50%), bladder cancer (40%, see Alcishi et al., Int. J. One. 4 (1994) 85) or lung carcinoma and myeloid leukemia (30%, Bos, JL Cancer Res. 49 (1989) 4682).

The present invention will be described in more detail with the following examples, which should be considered as illustrative and not limiting. Le2βnde of the figures

Figure 1: Position on their target (A) and efficiency of hybridization (B) of antisense 1-5, measured by the induction of a cut of target TARN for RNase H. Position on their target (C) and efficiency hybridization (D) of the R1-R8 antisense measured as in (B). Materials and methods

Synthesis of single-stranded antisense nucleic acids The various nucleic acids used were synthesized using an automatic solid phase synthesizer (Applied Biosystems, Forster city, CA) according to the chemistry of phosphoramidites. The nucleic acids were then precipitated twice with ethanol, washed in the presence of 75% ethanol, then dissolved in water. RNase H cleavage

The RNase H cleavage experiments from nuclear extracts of HeLa cells (HeLa Scribe, Nuclear extract, Promega) were carried out in the reaction mixture (25 μl) containing 40 mM Tris pH 7.9; 100 mM KC1; 2 raM MgC12; 0.8 μl of nuclear extract of HeLa, 2 μg of transporter DNA, 4 nM of wild or mutated ras mRNA and 5 μM of each nucleic acid to be tested. The reactions were carried out without premixing the mRNA and the nucleic acid to be tested. After treatment with phenol and precipitation, the cleavage products by RNase H were analyzed by electrophoresis on polycarylamide sequencing gel (6%). The gels were then autoradiographed, and the amount of intact material and cleavage product was determined by densitometry. Cell lines used

- HBL100: human epithelial cell line accessible to ATCC

- HBLlOOrasl: This line is derived from the human mammary line HBL100 by transformation with a plasmid pSV2 carrying the human Ha-Ras oncogene EJ / T24. This line expresses the wild form of Ha-ras, as well as the form carrying the point mutation G -> U in position 12, coding for the amino acid valine instead of a glycine (Ha-ras VAL12 (Lebeau et al. , Oncogene 6 (1991) 1125).

- HBL10OOneo: This line also derives from the human breast line HBL100, by transformation with a control plasmid pSV2. This line therefore expresses only the wild form of Ha-ras. Nanoparticle preparation

- Polyisohexylcyanoacrylate nanoparticles A nanoparticulate suspension was prepared by adding, with magnetic stirring, 100 μl of isobutylcyanoacrylate or isohexylcyanoacrylate to a polymerization medium composed of hydrochloric acid (1 mM, pH = 3 or 10 mM, pH = 2) and 1 g / 100 cm3 of dextran. The polymerization of cyanoacrylic monomers takes place spontaneously at a temperature in the region of 20 ° C. The polymerization is complete after 2 hours using isobutylcyanoacrylate and after 6 hours using isohexylcyanoacrylate. After neutralization, 0.4 g / 100 cm3 of poloxamer 188 is added to stabilize the nanoparticles. - Lactic acid-glycolic acid nanoparticles: 10 mg / cm3 of a lactic acid-glycolic acid copolymer containing 75% of lactic units D and L and 25% of glycolic units are suspended in a medium containing 10 mM TRIS-HC1 at pH = 7 and 0.5% lecithin. After 12 hours of incubation, the nanoparticles can be separated from the suspension. Encapsulation of nucleic acids in nanoparticles The nanoparticles prepared above at a concentration of 0.5 mg cm3 are incubated with 500 μM of nucleic acid, 500 μM of hydrophobic cation (for example CTAB), in a Tris-HCl buffer (10 mM, pH = 7 ) without sodium chloride (Chavany et al, Pharmaceutical Res. 9 (1992) 441). After 6 to 8 hours of incubation, the nanoparticles can be separated from the suspension or the suspension can be used, after dilution, to test the efficiency of the nucleic acid adsorbed on the appropriate cells.

Generally, a few μl of the diluted suspension (approximately 100 times in distilled water) are added to 100 μl of the culture medium containing the cells, the number of μl added being calculated to obtain the desired concentration of nucleic acid.

Examples

Example 1: Selection of the antisense 1.1. Effect of length

This example shows that length can have a significant impact on both the selectivity and the affinity of the antisense and that, contrary to the general trend, the use of antisense of limited size offers very important advantages. The following antisense have been synthesized:

- 5'-CACACCGACGGC (SEQ ID n ° 1) - 12 sea

- 5'-CACACCGACGGCG (SEQ ID n ° 2) - 13 sea

- 5'-CACACCGACGGCGC (SEQ ID n ° 3) - 14 sea

- 5'-CACACCGACGGCGCC (SEQ ID n ° 4) - 15 sea - 5'-CCACACCGACGGCGCC (SEQ ID n ° 5) - 16 sea

The position of these antisense vis-à-vis the target sequence (SEQ ID No. 6) is presented in Figure 1A. The corresponding antisense directed against the normal ras messenger were also synthesized. These different antisense were incubated in the presence of nuclear extracts of HeLa cells, and their ability to hybridize with the mutated ras messenger was determined by cleavage by RNase H as described in the materials and methods. The results obtained are shown in Figure IB. They show that the AS-Val antisense already produces a maximum response. They also show that an important non-selective signal appears as soon as the length of the antisense exceeds 13 nucleotides. These results therefore show that the best selectivity / affinity ratio is obtained with the antisense of 12 or 13 mer.

1.2. Position effect

This example shows that, in addition to the length, the position of the antisense relative to its target sequence is important.

The following 12-mer antisense have been synthesized:

- 5'-TGCCCACACCGA (RI: SEQ ED n ° 7)

- 5 * -GCCCACACCGAC (R2: SEQ ID n ° 8)

- 5'-CCACACCGACGG (R3: SEQ ID n ° 9) - 5 ^* -C ACACCGACGGC (R4: SEQ ID n ° 1)

- 5'-CACCGACGGCGC (R5: SEQ ID n ° 10)

- 5'-CCGACGGCGCCC (R6: SEQ ID n ° 11)

- 5'-GACGGCGCCCAC (R7: SEQ ID n ° 12)

- 5'-ACGGCGCCCACC (R8: SEQ ID n ° 13)

The position of these antisense vis-à-vis the target sequence (SEQ ID No. 6) is presented in Figure 1C. These different antisense were incubated in the presence of nuclear extracts of HeLa cells, and their capacity to hybridize with the mutated ras messenger was determined by cleavage by RNase H as described in the materials and methods. The results obtained are shown in Figure ID. They show that the antisense centered around the mutated region of the target sequence (R4 and R5) have the best selectivity towards RNA of the mutated oncogene, although they do not have the most efficacy. big. Example 2: Preparation of 5'-HO (CH- _ι: CACACCGACGGC (5'-m ₁₂ OH-AS2V 2.1) 1 -Dimethoxytrityl- 1, 12-dodecanediol (Dmt-O- (CH -) 12ÛH) To a solution of 1,12-dodecanediol (15 mmol) and N-ethyl-dimethylamine (6 mmol) in 20 cm 3 of anhydrous pyridine, dimethoxytrityl chloride (3 is added at a temperature of 20 ° C. mmoles) then continues stirring for 2 hours at a temperature in the region of 20 ° C. To the reaction mixture, 30 cm 3 of a 5% aqueous sodium hydrogencarbonate solution are added, then the product is extracted with dichloromethane. The organic phase is dried over sodium sulfate. After filtration and concentration to dryness under reduced pressure, the product obtained is purified by chromatography on silica gel, eluting with a dichloromethane-methanol mixture (97-3 by volume). There is thus obtained, with a yield of 75%, Dmt-O (CH2) i2θH, the characteristics of which are as follows: Rf = 0.65 (Kieselgel 60F 254 Merck; dichloromethane-methanol (9-1 by volume)

The compound Dmt-O- (CH ₂ -) ι ₂ OH obtained previously (1.2 mmol) is dried by co-evaporation under reduced pressure with anhydrous pyridine then it is kept under reduced pressure overnight. The product is dissolved in a solution of dimethylethylamine (0.44 g) in dichloromethane (8 cm3) then 2-cyanoethyl-N, N-dϋsopropylamino-chloro-phosphite is added slowly, under an argon atmosphere and with stirring (0.7 g, 3 mmol). After 30 minutes of reaction, 50 cm 3 of ethyl acetate are added to the reaction mixture, then the organic solution is washed with a 10% aqueous solution of sodium hydrogen carbonate (2 times

80 cm3) and finally with a saturated aqueous solution of sodium chloride (20 cm3).

The organic phase is dried over sodium sulfate. After filtration and concentration to dryness under reduced pressure, the product is chromatographed on a column of silica gel, eluting with a mixture of ethyl acetate and triethylamine. We thus obtain with a yield of 80% the OCH ₂ CH ₂ CN Dmt-O- (CH ₂ -) ι ₂ OP with the following characteristics:

N (iPr) ₂

Rf = 0.73 and 0.8 (stereoisomers) (Kieselgel 60F 254 Merck; ethyl acetate-triethylamine (9-1 by volume).

2.3) The assembly of the dodecadesoxynucleotide is carried out according to the phosphoramidite method on a DNA synthesizer using the deoxycytidine CPG (10 μmoles) and 50 μmoles of deoxyribonucleoside-3 '- (2-cyanoethyl) - dϋsopropylaminophosphoramidite per cycle. After detritylation, the support is treated for 10 minutes under an argon atmosphere with a solution of the phosphoramidite prepared according to Example 2.2 (1 cm3 0.1M in actonitrile) and tetrazole (3 cm3 0.5M in acetonitrile) . After elimination of the liquid phase, the support is then treated with 10 cm 3 of an iodine solution [0.01 M iodine in the acetonitrile-water-collidine mixture (65-30 by volume)]. The support is isolated and then treated with a concentrated ammonia solution for 7 hours at 55 ° C. After removing the support by filtration, the solution is evaporated to dryness under reduced pressure and then the residue is treated, for 30 minutes at a temperature in the region of 20 ° C, with acetic acid. The acetic acid is removed by evaporation under reduced pressure. The product obtained is purified by FPLC. The retention time of the purified product is given in Table I.

Example 3 - Preparation of 5'CACACCGACGGC- (CH _ι: OH f AS2-3'-m _I2 OH1 3-1) 1 -Dimethoxytrityl- 12-succinyl- 1, 12-dodecanediol

Dmt-O- (CH ₂ -) _{j 2} OC-CH ₂ -CH ₂ -COH

O O

A solution of the product prepared according to Example 2.1 (2 mmol), dimethylaminopyridine (1.1 mmol) and succinic anhydride (1) is reacted at a temperature in the region of 20 ° C. with stirring for 18 hours. , 6 mmol) in anhydrous pyridine (4 cm3). The pyridine is removed under reduced pressure. The residue obtained is taken up with 15 cm3 of dichloromethane then the organic solution is washed with a 10% aqueous citric acid solution (2 × 10 cm3) then with water. The organic phase is dried and evaporated. The residue which is dried under reduced pressure has the following characteristics:

Rf = 0.5 [Kieselgel 60F 254 Merck; dichloromethane-methanol (9-1 by volume)]. 2) Dmt-O- (CH ₂ -) ₁₂ OC-CH ₂ -CH ₂ -CNH (CH ₂ ) ₃ -Fractosil 500

OOA a solution of product prepared according to Example 3.1 (0.5 mmol) in a p.dioxane / pyridine mixture (95-5 by volume; 2.1 cm3) is added with stirring at a temperature in the region of 20 ° C, a solution of dicyclohexylcarbodiimide (1.25 mmol) in 300 μl of p.dioxane. Stirring is continued for one hour at a temperature in the region of 20 ° C., the dicyclohexylurea is removed by filtration. The solution obtained is treated with 1.5 g of aminopropyl-Fractosil 500 suspended in dimethylformamide and 0.38 cm3 of triethylamine for 18 hours at a temperature in the region of 20 ° C. The support is isolated by filtration and then washed with acetonitrile and dried under reduced pressure. The capacity of the support obtained is determined by spectrophotometric determination of the quantity of dimethoxytrityl cations released by an acid treatment of a sample of the dry support. In general, the support has a capacity of 70 μmoles g. In order to acetylate the residual amino functions, the support is taken up in 6 cm3 of pyridine and treated with 0.6 cm3 of acetic anhydride in the presence of 30 mg of 4-dimethylaminopyridine. After one hour of reaction, the support is isolated by filtration, washed with dichloromethane, methanol and ether and then dried under reduced pressure.

3-3) Using the support prepared according to example 3.2 (10 μmoles), the assembly of the dodecamer sequence is carried out automatically on a synthesizer according to the phosphoramidite method via deoxyribonucleo-side-3 ' - (2-cyanoethyl) -dϋsopropylaminophosphoramidite (50 μmoles per cycle). The deprotection and the purification of the dodecamer are carried out according to the conditions described in Example 2.3. The retention time of the purified product is given in Table I. Example 4 - Preparation of ⁵ '(CCACACCGA) ³ ' p (CH ₂ ) ι ₂ OH

By operating as in Example 3, the nonanucleotide was obtained (SEQ ID No. 14), the retention time of which is given in Table I. Example 5: Preparation of HO (CH ₂ ) ₁₂ pd ⁵ '(CACACCGACGGC) ³ ' p- (CH ₂ ) ι ₂ OH (5'ml2OH-AS2-3τnl2OF).

By operating as in Example 2 and replacing the CPG deoxycytidine with the support prepared according to Example 3, the dodecamer substituted at 3 'and at 5' by the group (CH ₂ ) ι OH is prepared. Table I gives the retention time of the purified product.

TABLE 1

Example Retention time

(minutes) *

2 20.4

• - 20.1

4 19.2

5 23.2

* FPLC: Mono P HR 5/5 column (Pharmacia); solvent A: 0.01 M NaH ₂ Pθ4 in the acetonitrile-water mixture (2-8 by volume; pH = 6.8) and solvent B: 0.01M NaH ₂ PO_ι, 1M NaCl in the acetonitrile-water mixture (2 -8 by volume; pH = 6.8); linear gradient from 0 to 100% in B; flow rate: 1 cm3 / minute.

Example 6: Preparation of d5 '(CACCGACGGCGC) p (CH ₂ ) ₃ OH

6.1) Dimethoxytrityl-1,3-propanediol

0.725 ml (10 mmol) of 1,3-propanediol was dissolved in 20 ml of anhydrous pyridine, and 4.07 g (12 mmol) of dimethoxytrityl chloride were added. The mixture is stirred for 2 h at room temperature, after which the TLC (CH2Cl2-MeOH 99: 1) shows that the reaction is practically complete. The reaction is then stopped by adding an excess of MeOH then, after 10 min, the mixture is concentrated. The concentrate is partitioned between the CH2C12 phase and a 5% aqueous NaHCO3 solution (2x), then the organic phase is dried over Na2SO4. Purification on gel of silica leads to 1.7 g (4.47 mmol) of the expected product. Rf = 0.44 (Alugram Sil G / UN254, Macherey Νagel, CH2C12-MeOH 99: 1).

6.2) Dimethoxytrityl-3-succinyl- 1, 3-propanediol

To 1.62 g of the product obtained above dissolved in 20 ml of anhydrous pyridine were added 1 g (10 mmol) of succinic anhydride and 1.22 g (100 mmol) of dimethylamino pyridine. The mixture is stirred for 16 h at room temperature, then an excess of water was added to stop the reaction. The mixture was concentrated and partitioned between a cold ethyl acetate phase and a 10% cold citric acid solution. The organic phase was washed once more with the cold citric acid solution and then with salt water. The organic phase is dried over Νa2SO4 and then purified on silica gel on a MeOH gradient (0 to 5%) in CH2Cl2 containing 0.25% of triethylamine, leading to 1.3 g of the expected product. Rf = 0.48 (Alugram Sil G / UV254, Macherey Nagel, CH2C12-MeOH 95: 5).

6.3) By operating as in Example 3 was obtained decanucleotide AS4- 3'-m3OH (SEQ ID ^No. 10).

Example 7: Preparation of d5 '(CACCGCCGGCGC) p (CH ₂ ) ₃ OH

By operating as in Example 6, the dodecanucleotide (SEQ

ID # 15). The sequence SEQ ID No. 15 is complementary to the normal ras mRNA.

Example 8: Encapsulation of antisense 8.1. The antisense adsorbed on the nanoparticles can be prepared in the following manner:

- A nanoparticulate suspension is prepared by adding, with magnetic stirring, 100 μl of isobutylcyanoacrylate or isohexylcyanoacrylate to a polymerization medium composed of hydrochloric acid (1 mM, pH = 3 or 10 mM, pH = 2) and 1 g / 100 cm3 of dextran. The polymerization of cyanoacrylic monomers takes place spontaneously at a temperature in the region of 20 ° C. The polymerization is complete after 2 hours using isobutylcyanoacrylate and after 6 hours using isohexylcyanoacrylate. After neutralization, 0.4 g / 100 cm3 of poloxamer 188 is added to stabilize the nanoparticles. - The nanoparticles thus obtained at a concentration of 0.5 mg / cm3 are incubated with 128 μM of modified antisense according to the invention, 500 μM of cation hydrophobic (for example CTAB), in a Tris-HCl buffer (10 mM, pH = 7) without sodium chloride. After 12 hours of incubation, the nanoparticles can be separated from the suspension or the suspension can be used, after dilution, to test the effectiveness of the antisense adsorbed on the appropriate cells. Generally, a few μl of the diluted suspension (approximately 100 times in distilled water) are added to 100 μl of the culture medium containing the cells, the number of μl added being calculated to obtain the desired concentration of antisense.

8.2. The antisense adsorbed on the nanoparticles can also be prepared in the following manner: 10 mg / cm 3 of a lactic acid-glycolic acid copolymer containing 75% of lactic units D and L and 25% of units are suspended glycolic in a medium containing 10 mM TRIS-HCl at pH = 7 and 0.5% lecithin. The nanoparticles are incubated with 2 μM of antisense, 1.6 mM of cetyltrimethylammonium bromide. After 12 hours of incubation, the nanoparticles can be separated from the suspension.

The rate of adhesion of the antisense to the nanoparticles is 82%.

EXAMPLE 9 Inhibition of the Growth of HBL10OOrasl Cells

This example demonstrates the quite remarkable inhibition properties of the antisense according to the invention.

The HBL10OOrasl cells were seeded in 96-well plates at a concentration of 4.10 ³ cells per well in 50 μl of medium supplemented with 7% heat-inactivated fetal calf serum, antibiotics and glutamine. When the cells adhere to the culture well (generally after 2 to 3 days), the antisense are added, to each well, at twice the final concentration in 50 μl of culture medium, resulting in a final volume of 100 μl per well. After 72 hours of incubation at 37 ° C in a humidified atmosphere containing 5% CO2, the cells were counted using a hemocytometer. Cell viability, examined at the same time by trypan blue staining, is greater than 95% for the treated and untreated cells. The results obtained are presented in Table 2 below. They are expressed as a percentage of proliferation of the cells relative to the untreated cells, according to the following formula: 100 x (Nn-NO) / (Nn-N), in which NO is the number of cells present at the start, Nn is the number of untreated cells after n days of growth, and N is the number of cells treated after n days.

TABLE 2

HBL 100-rasl cells HBL 100 cells

Product Concentration% Concentration% μM proliferation μM proliferation

AS4 (SEQ 10) 10 26 10 89

AS-GLY (SEQ 15) 20 86 20 95

AS2 (SEQ 1) 10 94 10 100

AS2 adsorbed 0.1 45 0.1 75

0.2 22 0.2 75

AS2-3'-mι ₂ OH 2 92

(Example 3)

adsorbed 0.05 32

AS4-3'-m ₃ OH 0.025 2 0.025 100

(Example 6) 0.05 16 0.05 100

0.005 40 0.005 100

AS-GLY-3'-m ₃ OH

(Example 7) 0.05 100

0.005 100 0.005 80

0.5 97 0.5 61 m3: - (CH ₂ ) ₃ - m ₁₂ : - (CH ₂ ) _Î2 - The results obtained show that:

- The antisense (AS4-3'm OH) modified according to the invention induces an inhibition of the proliferation of HBLlOOrasl cells at very low concentrations. These concentrations are approximately 100 times lower than those at which the unmodified antisense is active.

- The antisenses of the invention are specific for cells containing mutated ras since no effect is observed on HBL10OOneo cells. Similarly, the antisense directed against the normal gene (AS-Gly) has no effect on the proliferation of HBL10OOrasl cells.

These results therefore show that the compositions of the invention exhibit very high selectivity and activity. The maximum effect for a single injection of antisense was observed three days after adding the antisense to the culture.

Additional results have been obtained with another series of antisense (see Table 3). The results represent inhibition of cell proliferation

HBLlOOrasl cultured in the presence of the indicated antisense for 24 hours. The concentrations required to inhibit 50% of cell proliferation are given in Table 3.

These results confirm the very important selectivity and activity of the antisense of the invention. Particularly pronounced effects are obtained with 2,2-dimethyl-1,3 propanediol and with acridine-dodecanediol (Acr5'-AS4-3'ml2OH).

Example 12: Affinity study

This example shows that the antisense according to the invention have particularly high affinities for the target substrate substrate sequence. For this, the antisense of the invention AS4 (SEQ ID No. 10) modified at their 3 'end by different groups were used, including the antisense Acr5'-AS4-3'ml2OH. These antisense were placed in the presence of a ribonucleic target sequence, radiolabelled, with a length of 27 nucleotides. Late gel analyzes make it possible to determine the concentrations required to fix 50% of the substrate. The results obtained are presented in Table 4. Table 3

Dwarf X Formula 50% (μM) HBLlOOras cells Growth inhibition

R5 15

1, 3 Propanediol -0- (CH2) 3-OH 0.042

OH

1, 2 Propanediol -0- (CH2) -CH-CH3 2.5

CH3

2,2-Dimethyl-l, 3- Propanediol -O-CH2-C-CH2-OH 0.06

CH3

H

Ami no -Propanediol -O-CH2-C-CH2-NH2 0.150

OH

Hexanediol -0- (CH2) 6-OH 0.5

Decanediol -0- (CH2) ιo-OH 0.25

Dodecanedi ol -0- (CH2) i2-OH 0.06

Acridine- Acridine, - (CH2) 12-OH 0.04 Dodecanediol

Diethylenglycol -0- (CH2) 2-0- (CH2) 2OH 0.06

Glycerol -O-CH2-CHOH-CH2OH 0.04 Table 4

Name X Formula 50% (μM) Bindiπg

R5 0.19

1, 3 Propanediol -0- (CH2) 3-OH 0.15

OH

1, 2 Propanediol 1

-0- (CH2) -CH-CH3 0.2

CH3

2, 2-Dimethyl-l, 3- Propanediol 1 -0-CH2-C-CH2-0H 0.19

1

CH3

H

Amino-Propanediol -O-CH2-C-CH2-NH2

0.75 OH

Hexanediol -0- (CH2) 5-OH 0.3

Decanediol -0- (CH2) ιo-OH 0.6

Dodecanediol -0- (CH2) i2-OH 2,0

Acridine- Acridine, - (CH2) 12 -OH 0.7 Dodecanediol

SUBSTITUTE SHEET (RULE 26)

Claims

CLAIMS 1 - Single-stranded antisense nucleic acid complementary to a region of an mRNA of a human ras oncogene carrying a point mutation, characterized in that it comprises 8 to 20 nucleotides and a group of formula - (CHR) _n -OH in 3 ′ and / or in 5 ′, in which n represents an integer between 1 and 20 inclusive, and R represents a hydrogen atom or an OH group, R possibly varying from a group (CHR) to another.

2. Nucleic acid according to claim 1 characterized in that n represents an integer between 3 and 12 inclusive.

3. Nucleic acid according to claim 1 or 2 characterized in that the group of formula - (CHR) n-OH is chosen from dodecanediol, propanediol, dimethyl propanediol and glycerol.

4 - Nucleic acid according to one of claims 1 to 3 characterized in that it comprises from 10 to 13 nucleotides.

5 - Nucleic acid according to claim 4 characterized in that it comprises

12 nucleotides.

6. Nucleic acid according to one of claims 1 to 5 characterized in that the point mutation is located in the center of the complementary region.

7. Nucleic acid according to one of claims 1 to 6 characterized in that it is chosen from AS2-3'ml2OH; 5'ml2OH-AS2; AS2-3'm3OH; 5'm3OH-AS2;

5'ml2OH-AS2-3'ml2OH, AS4-3'ml2OH; 5'ml2OH-AS4; AS4-3'm3OH; 5 * m3OH- AS4 and 5'ml20H-AS4-3'ml20H, AS2-3 'Glycerol; AS4-3 'Glycerol.

8. Nucleic acid according to one of claims 1 to 7 characterized in that it comprises an additional chemical modification.

9. Nucleic acid according to claim 8 characterized in that it comprises one or more intercalating agents, such as acridine.

10. Nucleic acid according to claim 8 characterized in that it is the antisense of the antisense Acr5'-AS4-3'ml2OH

11. Nucleic acid according to one of claims 1 to 10 characterized in that it is encapsulated, included or adsorbed in a nanophase.

12 - Nucleic acid according to claim 1 1 characterized in that it is previously associated with cationic hydrophobic compounds.

13 - Pharmaceutical composition characterized in that it comprises one or more nucleic acids according to one of claims 1 to 12 in combination with one or more pharmaceutically acceptable diluents or adjuvants.

14. Pharmaceutical composition according to claim 13 intended for intratumoral administration.