WO1996018635A1 - Fullerene-oligonucleotide or fullerene-nucleotide conjugates, complexes thereof with nanoparticles, and therapeutical uses thereof - Google Patents

Fullerene-oligonucleotide or fullerene-nucleotide conjugates, complexes thereof with nanoparticles, and therapeutical uses thereof Download PDF

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Publication number
WO1996018635A1
WO1996018635A1 PCT/FR1995/001649 FR9501649W WO9618635A1 WO 1996018635 A1 WO1996018635 A1 WO 1996018635A1 FR 9501649 W FR9501649 W FR 9501649W WO 9618635 A1 WO9618635 A1 WO 9618635A1
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conjugate
fullerene
oligonucleotide
nucleotide
complex according
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PCT/FR1995/001649
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French (fr)
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Claude Helene
Alexandre Boutorine
Eichi Nakamura
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present invention relates to fullerene-oligonucleotide conjugates and fullerene-nucleotide conjugates as well as complexes comprising such conjugates associated with nanoparticles.
  • the oligonucleotides can be used against both single-stranded nucleic acids and messenger RNA or double-stranded DNA (Hélène and Toulmé, Biochim, Biophys. Acta, 1990, 1049, 99-1258); Thuong and Hélène, Angew, Chem. 1993, 105, 697-726; Angew. Chem. Int. Ed. Engl. 1993, 32, 666-690) to block the translation of the target messenger RNA (antisense strategy) or the transcription of the target gene by the formation of a triple helix (anti-gene strategy).
  • the present invention therefore relates to a fullerene-oligonucleotide or fullerene-nucleotide conjugate, in which said fullerene is advantageously a fullerene at Cg Q to Cg4 •
  • fullerenes can be in particular those described by Diederich and Rubin (1992 previously cited) and in particular a fullerene in Cg 0 , or a higher fullerene in C 7Q or in Cg 4 .
  • the oligonucleotide covalently linked to the fullerene in order to form the above-mentioned conjugate is advantageously a simple oligonucleotide strand and may comprise from 2 to 100 nucleotides.
  • Such oligonucleotides have a sequence complementary to that of the site in the DNA or RNA molecule into which the cleavage or cleavage is to be introduced, also called the target molecule.
  • target DNA or RNA can be, for example, sequences of viruses or sequences of genes involved in cancer and / or viral pathologies.
  • the above-mentioned conjugate can also be associated with a nanoparticle in the form of a complex, and in particular with a hydrophobic nanoparticle.
  • a nanoparticle is formed from a polylactic acid or from a polylactic acid-glycolic copolymer, optionally associated with polyethylene glycol, or from a polyalkylcyanoacrylate such as polyisohexylcyanoacrylate.
  • the conjugates not associated with a nanoparticle form structures observable by electron microscopy which can be filaments of the order of several tens of nanometers in length, and having a diameter of less than 10 nm, or nacelles of the order of 40 at 50 nm in diameter.
  • the filaments disappear and the conjugates associate with the nanoparticles.
  • a particular advantage of these complexes lies in the absence of hydrophobic cations such as cetyltrimethylammonium bromide (CTAB), considered to be cytotoxic.
  • CTAB cetyltrimethylammonium bromide
  • Another advantage of the association of these conjugates with hydrophobic nanoparticles lies in an improvement in the absorption of these conjugates on cell membranes, and therefore in an improvement in the penetration of these conjugates into living cells.
  • the conjugates according to the present invention can bind to various molecules, such as messenger and viral RNA (antisense and oligonucleotide clamp strategy), to DNA (triple helix strategy) or to proteins ( aptamer strategy).
  • messenger and viral RNA antisense and oligonucleotide clamp strategy
  • DNA triple helix strategy
  • proteins aptamer strategy
  • the fullerene derivative at Cg 0 which is advantageously used for the formation of conjugates according to the invention, has an absence of toxicity in mice, the lethal dose being greater than 500 mg / kg.
  • conjugates which are the subject of the present invention, and their complexes formed with nanoparticles can be used for therapeutic purposes, in order to totally or partially inhibit the expression of a given gene, for example by inhibiting the transcription of the gene or the translation of messenger RNA, either in the absence or in the presence of visible or ultraviolet light irradiation.
  • Such genes may in particular be genes responsible for or involved in pathologies of the cancer type, such as oncogenes, or genes of viruses such as the HIV virus.
  • the present invention due to the specificity of binding of the oligonucleotide has the advantage of being able to intervene very specifically in specific regions of genes whose sequence is at least partially known.
  • the present invention therefore relates to pharmaceutical compositions containing an effective amount of a conjugate or of a complex as described above, in combination with pharmacologically compatible excipients.
  • Such a composition will advantageously comprise from 1 ⁇ g to 10 mg of the conjugate which is the subject of the present invention.
  • the present invention further relates to a medicament containing such a conjugate or such a complex.
  • conjugates according to the present invention consists in the analysis of DNA fragments. To do this, the DNA preparations to be analyzed are placed in the presence of the conjugates, or their complexes, and are treated with low energy light, then the reaction products are demonstrated by methods known to man. of career.
  • the conjugates, or their complexes are administered by intravenous injection, by intradermal route or by local application.
  • the individual to whom these molecules have been administered can also be treated with low energy light.
  • the present invention also relates to a method for manufacturing a conjugate as described above, comprising activation of the fullerene and of the oligonucleotide, or of the nucleotide, allowing their covalent coupling.
  • a method comprises the following steps:
  • the final product is a conjugate having, in the case of a fullerene at Cg 0, the formula according to scheme III below.
  • FIGS. 1a to 1d respectively illustrate the conjugate used (FIG. 1a) and the target molecules hybridized to said conjugate, which are single-stranded DNA (FIG. 1d), a double stranded DNA (FIG. le) or a double stranded DNA having a loop (FIG. 1d).
  • the arrows represent the cut-off sites.
  • FIG. 2 is an electrophoresis gel of the structure having a loop, represented in FIG. 1d, placed in the presence of the conjugate represented on Figure la, without piperidine (wells 1 to 5) or in the presence of piperidine (wells 7 to 11), not irradiated
  • Wells 1 and 7 or irradiated with low energy light for 15 minutes (wells 2 and 8), 30 minutes (wells 3 and 9), 45 minutes (wells 4 and 10), and 60 minutes (wells 5 and 11 ).
  • the gel was analyzed using a phosphorimager.
  • Well 6 corresponds to the sequence of residues G obtained after treatment with dimethylsulfate and piperidine. The sequence in the cut-off region and the location of the guanine residues are indicated (the numbering starts from the 5 'end).
  • FIG. 3 is a graph in which the concentration in ⁇ M of oligo- fullerenes has been plotted on the abscissa and on the ordinate the percentage of adsorption, respectively without NaCl and with NaCl, in the case of the coupling of oligonucleotides - Fullerenes with polyalkylcyanoacrylate nanoparticles, in accordance with Example 3.
  • the yield of this reaction is of the order of 30 to 40%.
  • the conjugate has a brown-red color, is soluble in water and precipitates in ethanol or LiCl0 4 in acetone.
  • the three target molecules are: - an oligonucleotide of 20 nucleotides ( Figure lb); - a double stranded oligonucleotide of 26 base pairs ( Figure le); a oligonucleotide of 41 nucleotides partially double stranded and having a loop structure of five bases (FIG. 1d).
  • the complexes formed after the conjugate and the target molecules were irradiated using a Xenon lamp (1000 W) through a light absorbing filter at wavelengths less than 310 nm , in a buffer containing 10 mM cacodylate, pH 6, 50 mM NaCl, 50-100 nM of target molecule, with increasing concentrations of the conjugate (from 1 to 60 ⁇ M).
  • the three target molecules were labeled at the 5 ′ end with 32 P using gamma 32 P-ATP and polynucleotide kinase (marketed by Amersham).
  • FIG. 2 shows that the photo-cut induced by the conjugate takes place at well defined sites in the target molecules, contrary to the result obtained with unconjugated C 0 carboxylic acids, described above. It is observed that cuts take place mainly on guanines but also on thymines, although with lower efficiency.
  • the piperidine treatment slightly increases the intensity of the gel cut bands.
  • oligonucleotides to very hydrophobic groups, such as fullerenes C60 which makes it possible to increase their intracellular penetration.
  • Observations of fullerene oligonucleotides by transmission electron microscopy have shown that they are organized in the form of micelles 30 to 50 nm in diameter.
  • the adsorption of fullerene oligonucleotides to polyalkylcyanoacrylate nanoparticles was carried out. The objective was to combine these oligonucleotides chemically modified to nanoparticles, via hydrophobic interactions with the polymer matrix, without involving hydrophobic cations.

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Abstract

Conjugates of C60-84 fullerenes with oligonucleotides or nucleotides, and complexes of said conjugates with nanoparticles, e.g. polyalkylcyanoacrylate particles, are disclosed. Said conjugates and complexes may be used as drugs, in particular for treating cancerous and viral diseases.

Description

Conjugués fullérène-olioonucléotide, ou fullérène- nucléotide, leurs complexes avec des nanoparticules et leurs utilisations thérapeutiques.Fullerene-olioonucleotide, or fullerene-nucleotide conjugates, their complexes with nanoparticles and their therapeutic uses.
La présente invention a pour objet des conjugués fullérène-oligonucléotide et des conjugués fullérène-nucléotide ainsi que des complexes comprenant de tels conjugués associés à des nanoparticules.The present invention relates to fullerene-oligonucleotide conjugates and fullerene-nucleotide conjugates as well as complexes comprising such conjugates associated with nanoparticles.
Elle est en outre relative à l'utilisation thérapeutique de ces conjugués et complexes.It also relates to the therapeutic use of these conjugates and complexes.
Les oligonucleotides peuvent être utilisés aussi bien à l'encontre d'acides nucléiques simple brin que d'ARN messager ou d'ADN double brin (Hélène et Toulmé, Biochim, Biophys. Acta, 1990, 1049, 99- 1258); Thuong et Hélène, Angew, Chem. 1993, 105, 697- 726; Angew. Chem. Int. Ed. Engl. 1993, 32, 666-690) pour bloquer la traduction de l'ARN messager cible (stratégie anti-sens) ou la transcription du gène cible par la formation d'une triple hélice (stratégie anti-gène) .The oligonucleotides can be used against both single-stranded nucleic acids and messenger RNA or double-stranded DNA (Hélène and Toulmé, Biochim, Biophys. Acta, 1990, 1049, 99-1258); Thuong and Hélène, Angew, Chem. 1993, 105, 697-726; Angew. Chem. Int. Ed. Engl. 1993, 32, 666-690) to block the translation of the target messenger RNA (antisense strategy) or the transcription of the target gene by the formation of a triple helix (anti-gene strategy).
La substitution d'oligonucleotides par des agents réactifs peut induire des réactions irréversibles sur la séquence cible. Des substituants hydrophobes peuvent aussi être utilisés pour améliorer la fixation dans des systèmes biologiques (Boutourine et al. Biochimie, 1992, 74, 485-489; Krieg et al, Proc. Natl. Acad. Sci, USA 1993, 90, 1048-1052).Substitution of oligonucleotides by reactive agents can induce irreversible reactions on the target sequence. Hydrophobic substituents can also be used to improve binding in biological systems (Boutourine et al. Biochemistry, 1992, 74, 485-489; Krieg et al, Proc. Natl. Acad. Sci, USA 1993, 90, 1048-1052 ).
Des études récentes sur la chimie des fullerenes ont permis d'obtenir des fullerenes fonctionnalisés (Diederich et Rubin, Angew. Chem. 1992, 104, 1123-1146; Angew. Chem. Int. Ed. Engl.Recent studies on the chemistry of fullerenes have made it possible to obtain functionalized fullerenes (Diederich and Rubin, Angew. Chem. 1992, 104, 1123-1146; Angew. Chem. Int. Ed. Engl.
1992, 31, 1101-1123; Tokuyama et al, J. Ain. Chem. Soc.1992, 31, 1101-1123; Tokuyama et al, J. Ain. Chem. Soc.
1993, 115, 7918-7919).1993, 115, 7918-7919).
Des publications (Friedman et al; J. Am. Chem. Soc. 1993, 115, 6506-6509; Shinazi et al., Antimier, Agents Chemothr. 1993, 37, 1707-1710) ont montré que des fullerenes modifiés de manière adéquate ont une activité biologique sur des cellules vivantes, des enzymes, des virus et de l'ADN. Ainsi, l'irradiation d'un mélange d'ADN et du fullerène Cg0 lié à un acide carboxylique, par une lumière à faible énergie induit des clivages spécifiques au niveau des guanines sur les brins d'ADN. Cette sélectivité de clivage a été attribuée à l'action d'une espèce activée de l'oxygène générée par la photoactivation de la molécule de fullerène. Des expériences récentes (Tokuyama et Nakamura, J. Org. Chem. 1994, 59, 1135-1138) ont mis en évidence que l'acide carboxylique en Cg0 génère ces espèces activées de l'oxygène dans l'eau.Publications (Friedman et al; J. Am. Chem. Soc. 1993, 115, 6506-6509; Shinazi et al., Antimier, Agents Chemothr. 1993, 37, 1707-1710) have shown that adequately modified fullerenes have biological activity on living cells, enzymes, viruses and DNA. Thus, the irradiation of a mixture of DNA and of fullerene Cg 0 linked to a carboxylic acid, by low energy light induces specific cleavages at the level of the guanines on the DNA strands. This cleavage selectivity has been attributed to the action of an activated oxygen species generated by the photoactivation of the fullerene molecule. Recent experiments (Tokuyama and Nakamura, J. Org. Chem. 1994, 59, 1135-1138) have demonstrated that the Cg 0 carboxylic acid generates these activated species of oxygen in water.
Néanmoins, les méthodes décrites dans l'état de la technique ne permettent pas de cliver en un site spécifique une molécule d'ADN.However, the methods described in the prior art do not make it possible to cleave a DNA molecule at a specific site.
Le demandeur s'est donc attaché à résoudre ce problème.The applicant therefore set out to resolve this problem.
Il a trouvé que le couplage d'un fullerène avec un oligonucléotide, spécifique du site dans lequel doit être introduit le clivage, aboutit à un clivage spécifique de ce site. La présente invention a donc pour objet un conjugué fullérène-oligonucléotide ou fullérène- nucléotide, dans lequel ledit fullerène est avantageusement un fullerène en CgQ à Cg4 •He found that the coupling of a fullerene with an oligonucleotide, specific to the site into which the cleavage must be introduced, results in a specific cleavage of this site. The present invention therefore relates to a fullerene-oligonucleotide or fullerene-nucleotide conjugate, in which said fullerene is advantageously a fullerene at Cg Q to Cg4 •
De tels fullerenes peuvent être en particulier ceux décrits par Diederich et Rubin (1992 précédemment cités) et en particulier un fullerène en Cg0, ou un fullerène supérieur en C7Q ou en Cg4.Such fullerenes can be in particular those described by Diederich and Rubin (1992 previously cited) and in particular a fullerene in Cg 0 , or a higher fullerene in C 7Q or in Cg 4 .
L'oligonucléotide lié de manière covalente au fullerène afin de former le conjugué mentionné ci- dessus, est avantageusement un oligonucléotide simple brin et peut comprendre de 2 à 100 nucleotides.The oligonucleotide covalently linked to the fullerene in order to form the above-mentioned conjugate is advantageously a simple oligonucleotide strand and may comprise from 2 to 100 nucleotides.
De tels oligonucleotides présentent une séquence complémentaire de celle du site dans la molécule d'ADN ou d'ARN dans lequel doit être introduit la coupure ou le clivage, appelée aussi molécule cible.Such oligonucleotides have a sequence complementary to that of the site in the DNA or RNA molecule into which the cleavage or cleavage is to be introduced, also called the target molecule.
De tels ADN ou ARN cibles peuvent être par exemple des séquences de virus ou des séquences de gènes intervenant dans des pathologies cancéreuses et/ou virales.Such target DNA or RNA can be, for example, sequences of viruses or sequences of genes involved in cancer and / or viral pathologies.
Le conjugué mentionné ci-dessus peut aussi être associé à une nanoparticule sous la forme d'un complexe, et en particulier à une nanoparticule hydrophobe. Avantageusement, une telle nanoparticule est formée d'un acide polylactique ou d'un copolymère acide polylactique-glycolique, éventuellement associés à du polyéthylène glycol, ou d'un polyalkylcyanoacrylate tel que le polyisohexylcyano- acrylate.The above-mentioned conjugate can also be associated with a nanoparticle in the form of a complex, and in particular with a hydrophobic nanoparticle. Advantageously, such a nanoparticle is formed from a polylactic acid or from a polylactic acid-glycolic copolymer, optionally associated with polyethylene glycol, or from a polyalkylcyanoacrylate such as polyisohexylcyanoacrylate.
Ces dernières particules sont décrites par Chavany et al, (Pharmaceutical Research, 1992, 9, 441. )These latter particles are described by Chavany et al, (Pharmaceutical Research, 1992, 9, 441.)
Les conjugués non associés à une nanoparticule, forment des structures observables en microscopie électronique qui peuvent être des filaments de l'ordre de plusieurs dizaines de nanometres de longueur, et présentant un diamètre inférieur à 10 nm, ou des nacelles de l'ordre de 40 à 50 nm de diamètre. En présence de nanoparticules de polyisohexyl- cyanoacrylate, les filaments disparaissent et les conjugués s'associent aux nanoparticules. Un avantage particulier de ces complexes réside en l'absence de cations hydrophobes comme le bromure de cétyltriméthylammonium (CTAB) , considérés comme cytotoxiques .The conjugates not associated with a nanoparticle, form structures observable by electron microscopy which can be filaments of the order of several tens of nanometers in length, and having a diameter of less than 10 nm, or nacelles of the order of 40 at 50 nm in diameter. In the presence of polyisohexyl cyanoacrylate nanoparticles, the filaments disappear and the conjugates associate with the nanoparticles. A particular advantage of these complexes lies in the absence of hydrophobic cations such as cetyltrimethylammonium bromide (CTAB), considered to be cytotoxic.
Un autre avantage de l'association de ces conjugués à des nanoparticules hydrophobes réside dans une amélioration de l'absorption de ces conjugués sur les membranes cellulaires, et de ce fait dans une amélioration de la pénétration de ces conjugués dans les cellules vivantes.Another advantage of the association of these conjugates with hydrophobic nanoparticles lies in an improvement in the absorption of these conjugates on cell membranes, and therefore in an improvement in the penetration of these conjugates into living cells.
Les conjugués selon la présente invention, seuls ou associés sous forme de complexes, peuvent se fixer sur diverses molécules, telles que des ARN messager et viraux (stratégie antisens et pince oligonucléotidique) , sur des ADN (stratégie triple hélice ) ou sur des protéines (stratégie aptamères).The conjugates according to the present invention, alone or combined in the form of complexes, can bind to various molecules, such as messenger and viral RNA (antisense and oligonucleotide clamp strategy), to DNA (triple helix strategy) or to proteins ( aptamer strategy).
On notera en outre que le dérivé du fullerène en Cg0, qui est avantageusement utilisé pour la formation de conjugués selon l'invention, présente une absence de toxicité chez la souris, la dose létale étant supérieure à 500 mg/kg.It will also be noted that the fullerene derivative at Cg 0 , which is advantageously used for the formation of conjugates according to the invention, has an absence of toxicity in mice, the lethal dose being greater than 500 mg / kg.
Les conjugués objet de la présente invention, et leurs complexes formés avec des nanoparticules, peuvent être utilisés à des fins thérapeutiques, afin d'inhiber totalement ou partiellement l'expression d'un gène donné, par exemple en inhibant la transcription du gène ou la traduction de l'ARN messager, soit en l'absence soit en présence d'irradiation par la lumière visible ou ultraviolette.The conjugates which are the subject of the present invention, and their complexes formed with nanoparticles, can be used for therapeutic purposes, in order to totally or partially inhibit the expression of a given gene, for example by inhibiting the transcription of the gene or the translation of messenger RNA, either in the absence or in the presence of visible or ultraviolet light irradiation.
De tels gènes peuvent être en particulier des gènes responsables ou intervenant dans des pathologies du type cancer, tels que les oncogènes, ou des gènes de virus tels que le virus HIV.Such genes may in particular be genes responsible for or involved in pathologies of the cancer type, such as oncogenes, or genes of viruses such as the HIV virus.
La présente invention du fait de la spécificité de fixation de 1 ' oligonucléotide présente l'avantage de pouvoir intervenir de manière très spécifique dans des régions précises de gènes dont on connaît au moins partiellement la séquence. La présente invention a donc pour objet des compositions pharmaceutiques contenant une quantité efficace d'un conjugué ou d'un complexe tel que décrit ci-dessus, en association avec des excipients pharmacologiquement compatibles. Une telle composition comprendra avantageusement de 1 μg à 10 mg du conjugué objet de la présente invention.The present invention due to the specificity of binding of the oligonucleotide has the advantage of being able to intervene very specifically in specific regions of genes whose sequence is at least partially known. The present invention therefore relates to pharmaceutical compositions containing an effective amount of a conjugate or of a complex as described above, in combination with pharmacologically compatible excipients. Such a composition will advantageously comprise from 1 μg to 10 mg of the conjugate which is the subject of the present invention.
La présente invention est en outre relative à un médicament contenant un tel conjugué ou un tel complexe.The present invention further relates to a medicament containing such a conjugate or such a complex.
Elle a de plus pour objet l'utilisation d'un tel conjugué ou d'un tel complexe pour la fabrication d'un médicament pour le traitement de pathologies comme le cancer, ou pour le traitement de maladies virales.It further relates to the use of such a conjugate or such a complex for the manufacture of a medicament for the treatment of pathologies such as cancer, or for the treatment of viral diseases.
Une autre utilisation des conjugués selon la présente invention consiste en l'analyse de fragments d'ADN. Pour ce faire, les préparations d'ADN à analyser sont mises en présence des conjugués, ou de leurs complexes, et sont traités avec une lumière à faible énergie, puis les produits de réaction sont mis en évidence par des méthodes connues de l'homme du métier.Another use of the conjugates according to the present invention consists in the analysis of DNA fragments. To do this, the DNA preparations to be analyzed are placed in the presence of the conjugates, or their complexes, and are treated with low energy light, then the reaction products are demonstrated by methods known to man. of career.
Avantageusement, les conjugués, ou leurs complexes, sont administrés par injection intraveineuse, par voie intradermique ou par application locale. L'individu auquel a été administré ces molécules peut en outre être traité par une lumière à faible énergie. La présente invention est en outre relative à un procédé de fabrication d'un conjugué tel que décrit ci-dessus, comprenant une activation du fullerène et de l'oligonucléotide, ou du nucléotide, permettant leur couplage covalent. Avantageusement, un tel procédé comprend les étapes suivantes:Advantageously, the conjugates, or their complexes, are administered by intravenous injection, by intradermal route or by local application. The individual to whom these molecules have been administered can also be treated with low energy light. The present invention also relates to a method for manufacturing a conjugate as described above, comprising activation of the fullerene and of the oligonucleotide, or of the nucleotide, allowing their covalent coupling. Advantageously, such a method comprises the following steps:
- réaction d'un dérivé du fullerène comportant un groupe hydroxyle et de bromure de bromoacétate selon le schéma I ci-après, - introduction d'un groupement thiol sur un oligonucléotide ou un nucleotide non bloqué par réaction dudit oligonucléotide ou nucleotide avec de la N-méthylimidazole puis avec de la cystamine et création du groupe thiol par réaction du produit ainsi obtenu avec du dithiotreitol, selon le schéma II ci- après dans lequel Nu représente un nucleotide,- reaction of a fullerene derivative comprising a hydroxyl group and bromoacetate bromide according to scheme I below, - introduction of a thiol group on an oligonucleotide or an unblocked nucleotide by reaction of said oligonucleotide or nucleotide with N -methylimidazole then with cystamine and creation of the thiol group by reaction of the product thus obtained with dithiotreitol, according to scheme II below in which Nu represents a nucleotide,
- mise en contact des produits des deux étapes précédentes en présence de pyridine.- bringing the products of the two preceding stages into contact in the presence of pyridine.
Le produit final est un conjugué présentant, dans le cas d'un fullerène en Cg0 la formule selon le schéma III ci-après.The final product is a conjugate having, in the case of a fullerene at Cg 0, the formula according to scheme III below.
L'homme du métier se reportera avantageusement au manuel: Maniatis et al. 1982 Molecular Cloning- A Laboratory Manual- CSH éd. New York ou à l'une de ses récentes rééditions pour la mise en oeuvre de la présente invention.Those skilled in the art will advantageously refer to the manual: Maniatis et al. 1982 Molecular Cloning- A Laboratory Manual- CSH ed. New York or one of its recent reissues for the implementation of the present invention.
La présente invention est illustrée sans pour autant être limitée par les exemples suivants dans lesquels : Les figures la à ld illustrent respectivement le conjugué utilisé (figure la) et les molécules cibles hybridées audit conjugué, qui sont un ADN simple brin (figure ld), un ADN double brin (figure le) ou un ADN double brin présentant une boucle (figure ld) . Sur les figures lb à ld, les flèches représentent les sites de coupure.The present invention is illustrated without however being limited by the following examples in which: FIGS. 1a to 1d respectively illustrate the conjugate used (FIG. 1a) and the target molecules hybridized to said conjugate, which are single-stranded DNA (FIG. 1d), a double stranded DNA (FIG. le) or a double stranded DNA having a loop (FIG. 1d). In Figures 1b to 1d, the arrows represent the cut-off sites.
Le résultat de ces coupures est mis en évidence sur la figure 2 qui est un gel 'électrophorèse de la structure présentant une boucle, représentée sur la figure ld, mis en présence du conjugué représenté sur la figure la, sans piperidine ( puits 1 à 5) ou en présence de piperidine (puits 7 à 11), non irradiéThe result of these cuts is highlighted in FIG. 2 which is an electrophoresis gel of the structure having a loop, represented in FIG. 1d, placed in the presence of the conjugate represented on Figure la, without piperidine (wells 1 to 5) or in the presence of piperidine (wells 7 to 11), not irradiated
(puits 1 et 7) ou irradié par une lumière à faible énergie durant 15 minutes (puits 2 et 8), 30 minutes (puits 3 et 9), 45 minutes (puits 4 et 10), et 60 minutes (puits 5 et 11). Le gel a été analysé à l'aide d'un phosphor- imageur. Le puits 6 correspond à la séquence des résidus G obtenue après traitement avec le diméthylsulfate et la piperidine. La séquence dans la région de coupure et la localisation des résidus guanine sont indiquées (la numérotation débute à partir de l'extrémité 5' ) .(wells 1 and 7) or irradiated with low energy light for 15 minutes (wells 2 and 8), 30 minutes (wells 3 and 9), 45 minutes (wells 4 and 10), and 60 minutes (wells 5 and 11 ). The gel was analyzed using a phosphorimager. Well 6 corresponds to the sequence of residues G obtained after treatment with dimethylsulfate and piperidine. The sequence in the cut-off region and the location of the guanine residues are indicated (the numbering starts from the 5 'end).
La figure 3 est un graphique dans lequel on a porté en abscisses la concentration en μM d'oligo- fullerenes à l'équilibre et en ordonnées le pourcentage d'adsorption, respectivement sans NaCl et avec NaCl, dans le cas du couplage d'oligonucléotides- fullérènes avec des nanoparticules de polyalkylcyanoacrylates, conformément à l'exemple 3. EXEMPLE 1 -FIG. 3 is a graph in which the concentration in μM of oligo- fullerenes has been plotted on the abscissa and on the ordinate the percentage of adsorption, respectively without NaCl and with NaCl, in the case of the coupling of oligonucleotides - Fullerenes with polyalkylcyanoacrylate nanoparticles, in accordance with Example 3. EXAMPLE 1 -
Préparation d'un conjugué fullerène en C6Q- oligonucléotide. l') Formation d'un groupement réactif sur un méthanofullérène Un dérivé du fullerène tel que décrit parPreparation of a fullerene conjugate in C6Q- oligonucleotide. l ') Formation of a reactive group on a methanofullerene A derivative of fullerene as described by
Diederich et Rubin (1992, précédemment cités) et Tokuyama et al. (1993, précédemment cités) est mis à réagir avec du bromure de bromoacetate en présence de pyridine, dans du toluène à 20"C durant vingt minutes. 2') Préparation de l'oligonucléotide.Diederich and Rubin (1992, previously cited) and Tokuyama et al. (1993, previously cited) is reacted with bromoacetate bromide in the presence of pyridine, in toluene at 20 "C for twenty minutes. 2 ') Preparation of the oligonucleotide.
Un oligonucléotide non bloqué est mis à réagir avec de la N-méthylimidazole en présence de triphénylphosphine et de bis(2-thiopyridine) dans le diméthylsulfoxide ou le diméthylformamide à 20"C. Le produit obtenu est mis en présence de cystamine pendant 20 minutes puis de dithiotréitol afin d'obtenir un dérivé thiol.An unblocked oligonucleotide is reacted with N-methylimidazole in the presence of triphenylphosphine and bis (2-thiopyridine) in dimethylsulfoxide or dimethylformamide at 20 ° C. The product obtained is brought into contact with cystamine for 20 minutes then dithiotreitol to obtain a thiol derivative.
3*) Fabrication du conjugué.3 * ) Manufacturing of the conjugate.
Les produits des deux étapes précédentes sont mis à réagir en présence de triethylamine dans de la pyridine à 20°C.The products of the two preceding stages are reacted in the presence of triethylamine in pyridine at 20 ° C.
Le rendement de cette réaction est de l'ordre de 30 à 40%.The yield of this reaction is of the order of 30 to 40%.
Le conjugué présente une couleur marron-rouge, est soluble dans l'eau et précipite dans de 1 'éthanol ou du LiCl04 dans de l'acétone.The conjugate has a brown-red color, is soluble in water and precipitates in ethanol or LiCl0 4 in acetone.
La purification du conjugué par CHLP n'est pas possible en raison de l'adsorption irréversible du conjugué sur les supports de gel de silice modifié. Sur des gels de polyacrylamide le conjugué forme des agrégats dans les puits. L'électrophorèse sur des gels d'agarose à 1% comprenant du Triton X100 à 0,1 % révèle une bande homogène colorée migrant plus lentement que les oligonucleotides non conjugués. Cette technique a été utilisée pour purifier le conjugué des oligonucleotides n'ayant pas réagi. EXEMPLE 2 -Purification of the conjugate by CHLP is not possible due to the irreversible adsorption of the conjugate on the modified silica gel supports. On polyacrylamide gels the conjugate forms aggregates in the wells. Electrophoresis on 1% agarose gels comprising 0.1% Triton X100 reveals a homogeneous colored band migrating more slowly than the unconjugated oligonucleotides. This technique was used to purify the conjugate from unreacted oligonucleotides. EXAMPLE 2 -
Coupure de diverses molécules cibles d'ADN par le conjugué synthétisé dans l'exemple 1. Trois oligodésoxynucléotides différents ont été utilisés comme cible afin de tester les propriétés de coupure en présence de lumière d'un conjugué obtenu selon l'exemple 1 et comprenant un fullerène en Cg0 conjugué à un oligonucléotide de 14 nucleotides. Une représentation schématique de ce conjugué est celle de la figure la, sur laquelle la séquence de 1 ' oligonucléotide est représentée.Cleavage of various DNA target molecules by the conjugate synthesized in Example 1. Three different oligodeoxynucleotides were used as target in order to test the cleavage properties in the presence of light of a conjugate obtained according to Example 1 and comprising a fullerene at Cg 0 conjugated to a 14 nucleotide oligonucleotide. A schematic representation of this conjugate is that of FIG. La, in which the sequence of the oligonucleotide is represented.
Les trois molécules cibles sont: - un oligonucléotide de 20 nucleotides (figure lb); - un oligonucléotide double brin de 26 paires de bases (figure le); un oligonucléotide de 41 nucleotides partiellement double brin et présentant une structure en boucle de cinq bases (figure ld) .The three target molecules are: - an oligonucleotide of 20 nucleotides (Figure lb); - a double stranded oligonucleotide of 26 base pairs (Figure le); a oligonucleotide of 41 nucleotides partially double stranded and having a loop structure of five bases (FIG. 1d).
L'irradiation des complexes formés après mise en présence du conjugué et des molécules cibles a été effectuée à l'aide d'une lampe à Xénon (1000 W) à travers un filtre absorbant la lumière à des longueurs d'ondes inférieures à 310 nm, dans un tampon contenant 10 mM cacodylate, pH 6, 50 mM NaCl, 50-100 nM de molécule cible, avec des concentrations croissantes du conjugué ( de 1 à 60 μM) .The complexes formed after the conjugate and the target molecules were irradiated using a Xenon lamp (1000 W) through a light absorbing filter at wavelengths less than 310 nm , in a buffer containing 10 mM cacodylate, pH 6, 50 mM NaCl, 50-100 nM of target molecule, with increasing concentrations of the conjugate (from 1 to 60 μM).
Les trois molécules cibles ont été marquées à l'extrémité 5' par 32P à l'aide de gamma32P-ATP et de polynucléotide kinase ( commercialisée par Amersham) .The three target molecules were labeled at the 5 ′ end with 32 P using gamma 32 P-ATP and polynucleotide kinase (marketed by Amersham).
L'analyse par électrophorèse a été effectuée sur un gel de polyacrylamide à 12 % .dénaturant, avant et après le traitement par la piperidine. La figure 2 montre que la photo-coupure induite par le conjugué a lieu à des sites bien définis dans les molécules cibles, contrairement au résultat obtenu avec des acides carboxyliques en C 0 non conjugués, décrits précédemment. On observe que des coupures ont lieu principalement sur les guanines mais aussi sur les thymines, bien qu'avec une efficacité plus faible. Le traitement à la piperidine augmente légèrement l'intensité des bandes de coupure du gel.The electrophoresis analysis was carried out on a 12% denaturing polyacrylamide gel, before and after treatment with piperidine. FIG. 2 shows that the photo-cut induced by the conjugate takes place at well defined sites in the target molecules, contrary to the result obtained with unconjugated C 0 carboxylic acids, described above. It is observed that cuts take place mainly on guanines but also on thymines, although with lower efficiency. The piperidine treatment slightly increases the intensity of the gel cut bands.
La comparaison des bandes générées après traitement à la piperidine après la réaction avec le diméthylsulfate (DMS) suggère que les fragments obtenus par coupure photo-induite se termine par un 3' phosphate.The comparison of the bands generated after treatment with piperidine after the reaction with dimethylsulfate (DMS) suggests that the fragments obtained by photo-induced cleavage ends in a 3 ′ phosphate.
La localisation des sites de coupure sur les trois molécules cibles est représentée sur les figures lb , le et ld .The location of the cleavage sites on the three target molecules is shown in the figures lb, le and ld.
Les guanines les plus réactives sont localisées dans la région en forme de boucle de la structure tige-boucle (figure ld) . Sur l'ADN double brin, les coupures ont lieu sur les guanines du brin contenant des purines, à proximité de l'extrémité 3' du troisième brin de la structure formée (figure le) .The most reactive guanines are located in the loop-shaped region of the stem-loop structure (Figure 1d). On double-stranded DNA, the cuts take place on the guanines of the strand containing purines, near the 3 ′ end of the third strand of the structure formed (FIG. Le).
Aucune coupure n'est observée sur l'autre brin du double brin, qui ne contient aucune guanine, à proximité de l'extrémité 3' du troisième brin.No cut is observed on the other strand of the double strand, which contains no guanine, near the 3 'end of the third strand.
Sur la molécule cible simple brin ( figure lb) des coupures photo-induites ont lieu sur des guanines à proximité de l'extrémité 3' du conjugué formé. Les coupures ont lieu quasi exclusivement sur des bases guanines, ce qui est en accord avec l'hypothèse d'une intervention d'une espèce activée de 1 'oxygène.On the single-stranded target molecule (FIG. 1b) photo-induced cleavages take place on guanines near the 3 ′ end of the conjugate formed. The cuts take place almost exclusively on guanine bases, which is in agreement with the hypothesis of an intervention of an activated species of oxygen.
Les résultats indiquent que la partie fullerène du conjugué n'empêche pas la formation de structure double brin ou triple brin.The results indicate that the fullerene part of the conjugate does not prevent the formation of double strand or triple strand structure.
En outre, ils montrent que le conjugué oligonucléotide-fullérène se fixe spécifiquement sur la séquence cible en simple ou en double brin. EXEMPLE 3:In addition, they show that the oligonucleotide-fullerene conjugate specifically binds to the target sequence in single or double strand. EXAMPLE 3:
Cet exemple illustre le couplage d'oligonucleotides à des groupements très hydrophobes, tels que les fullerenes C60 qui permet d'accroître leur pénétration intracellulaire. Les observations des oligonucléotides-fullérènes en microscopie électronique à transmission ont montré qu'ils s'organisaient sous forme de micelles de 30 à 50 nm de diamètre. L'adsorption des oligonucléotides-fullérènes aux nanoparticules de polyalkylcyanoacrylates a été réalisée. L'objectif était d'associer ces oligonucleotides chimiquement modifiés aux nanoparticules, par l'intermédiaire d'interactions hydrophobes avec la matrice polymérique, sans faire intervenir de cations hydrophobes. Les isothermes d'adsorption des oligo- fullérènes (24 mère) aux nanoparticules (0,1 mg/ml) ont été réalisés en tampon Tris/HCl (10 mM) , dextran 70 (1%). Après 12 h d'adsorption, les nanoparticules ont été séparées des oligo-fullérènes non adsorbes par centrifugation (20 000 g/60 n) sur gradient de saccharose (20%). Les dosages ont été réalisés dans les surnageants, en mesurant les DO à 260 et 340 nm. L'adsorption est supérieure à 90 %, même en présence de NaCl pour des concentrations inférieures à lμm. Comme le montre la figure 3, qui est un graphique illustrant les résultats obtenus, les isothermes d'adsorption ont montré une importante adsorption, jusqu'à 90%, des oligonucléotides- fullérènes aux nanoparticules. Chez les deux types cellulaires étudiés HeLa et Ug37, l'incubation d'oligonucléotides-fullérènes jusqu'à la concentration de lOμM n'ayant pas montré de cytotoxicite permet donc de prévoir leur utilisation in vivo. This example illustrates the coupling of oligonucleotides to very hydrophobic groups, such as fullerenes C60 which makes it possible to increase their intracellular penetration. Observations of fullerene oligonucleotides by transmission electron microscopy have shown that they are organized in the form of micelles 30 to 50 nm in diameter. The adsorption of fullerene oligonucleotides to polyalkylcyanoacrylate nanoparticles was carried out. The objective was to combine these oligonucleotides chemically modified to nanoparticles, via hydrophobic interactions with the polymer matrix, without involving hydrophobic cations. Isothermal adsorption of oligo-fullerenes (24 mer) to nanoparticles (0.1 mg / ml) were carried out in Tris / HCl buffer (10 mM), dextran 70 (1%). After 12 h of adsorption, the nanoparticles were separated from the non-adsorbed oligo-fullerenes by centrifugation (20,000 g / 60 n) on a sucrose gradient (20%). The assays were carried out in the supernatants, by measuring the OD at 260 and 340 nm. The adsorption is greater than 90%, even in the presence of NaCl for concentrations below 1 μm. As shown in FIG. 3, which is a graph illustrating the results obtained, the adsorption isotherms have shown significant adsorption, up to 90%, of the fullerene oligonucleotides to the nanoparticles. In the two cell types studied HeLa and Ug37, the incubation of fullerene oligonucleotides up to the concentration of 10 μM which has not shown cytotoxicity therefore makes it possible to predict their use in vivo.
Figure imgf000014_0001
Figure imgf000014_0001
SCHEMA IDIAGRAM I
Figure imgf000014_0002
Figure imgf000014_0002
Figure imgf000014_0003
Figure imgf000014_0003
SCHEMA IIDIAGRAM II
Figure imgf000014_0004
Figure imgf000014_0004
SCHEMA IIIDIAGRAM III
DE REMPLACEMENT (RÈGLE 26) SUBSTITUTE (RULE 26)

Claims

REVENDICATIONS
1. Conjugué entre un fullerène et un oligonucléotide ou un nucleotide.1. Conjugate between a fullerene and an oligonucleotide or a nucleotide.
2. Conjugué selon la revendication 1, caractérisé en ce que ledit fullerène est en Cg0 à2. Conjugate according to claim 1, characterized in that said fullerene is in Cg 0 to
C84' C 84 '
3. Conjugué selon l'une des revendications 1 et3. Conjugate according to one of claims 1 and
2, caractérisé en ce que ledit fullerène est en CgQ.2, characterized in that said fullerene is in Cg Q.
4. Conjugué selon l'une des revendications 1 à 3, caractérisé en ce que ledit oligonucléotide est simple brin.4. Conjugate according to one of claims 1 to 3, characterized in that said oligonucleotide is single strand.
5. Conjugué selon l'une des revendications 1 à 4, caractérisé en ce que ledit oligonucléotide comprend de 2 à 100 nucleotides. 5. Conjugate according to one of claims 1 to 4, characterized in that said oligonucleotide comprises from 2 to 100 nucleotides.
6. Complexe comprenant un conjugué selon l'une des revendications 1 à 5 associé à une nanoparticule.6. Complex comprising a conjugate according to one of claims 1 to 5 associated with a nanoparticle.
7. Complexe selon la revendication 6, caractérisé en ce que ladite particule est hydrophobe.7. Complex according to claim 6, characterized in that said particle is hydrophobic.
8. Complexe selon l'une des revendications 6 et 7, caractérisé en ce que ladite particule est formée de polyalkylcyanoacrylate et avantageusement de polyisohexyl- cyanoacrylate.8. Complex according to one of claims 6 and 7, characterized in that said particle is formed of polyalkylcyanoacrylate and advantageously of polyisohexyl-cyanoacrylate.
9. Complexe selon l'une des revendications 6 et 7, caractérisé en ce que ladite particule est formée d'acide polylactique ou d'un copolymère acide polylactique-glycolique, éventuellement associés à du polyéthylène glycol.9. Complex according to one of claims 6 and 7, characterized in that said particle is formed of polylactic acid or of a polylactic-glycolic acid copolymer, optionally associated with polyethylene glycol.
10. Composition pharmaceutique contenant une quantité efficace d'un conjugué ou d'un complexe selon l'une des revendications 1 à 9 en association avec des excipients pharmacologiquement compatibles.10. Pharmaceutical composition containing an effective amount of a conjugate or of a complex according to one of claims 1 to 9 in combination with pharmacologically compatible excipients.
11. Médicament contenant un conjugué ou un complexe selon l'une des revendications 1 à 9.11. Medicament containing a conjugate or a complex according to one of claims 1 to 9.
12. Utilisation d'un conjugué ou d'un complexe selon l'une des revendications 1 à 9 pour la fabrication de médicaments pour le traitement des pathologies de type cancéreuses.12. Use of a conjugate or a complex according to one of claims 1 to 9 for the manufacture of drugs for the treatment of cancer-like pathologies.
13. Utilisation d'un conjugué ou d'un complexe selon l'une des revendications 1 à 9 pour la fabrication d'un médicament pour le traitement des maladies virales.13. Use of a conjugate or a complex according to one of claims 1 to 9 for the manufacture of a medicament for the treatment of viral diseases.
14. Procédé de fabrication d'un conjugué selon l'une des revendications 1 à 5, comprenant une activation du fullerène et de l'oligonucléotide, ou du nucleotide, permettant leur couplage covalent.14. Method for manufacturing a conjugate according to one of claims 1 to 5, comprising activation of the fullerene and of the oligonucleotide, or of the nucleotide, allowing their covalent coupling.
15. Procédé de fabrication d'un conjugué selon la revendication 14 comprenant les étapes suivantes:15. A method of manufacturing a conjugate according to claim 14 comprising the following steps:
- réaction d'un dérivé du fullerène comportant un groupe hydroxyle et de bromure de bromoacetate, - introduction d'un groupement thiol sur un oligonucléotide, ou un nucleotide, non bloqué par réaction dudit oligonucléotide, ou nucleotide, avec de la N-méthylimidazole puis avec de la cystamine et création du groupement thiol par réaction du produit ainsi obtenu avec du dithiotréitol, et- reaction of a fullerene derivative comprising a hydroxyl group and bromoacetate bromide, - introduction of a thiol group on an oligonucleotide, or a nucleotide, not blocked by reaction of said oligonucleotide, or nucleotide, with N-methylimidazole then with cystamine and creation of the thiol group by reaction of the product thus obtained with dithiotreitol, and
- mise en contact des produits des deux étapes précédentes en présence de pyridine. - bringing the products of the two preceding stages into contact in the presence of pyridine.
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WO2021162725A1 (en) * 2020-02-16 2021-08-19 Butzloff Peter Robert Pivoting electrodynamic composition and medicament

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