Title:  Conjugates of a particle vector and oligonucleotides, process for their preparation, and pharmaceutical compositions containing them

United States Patent:  6,214,621

Assignee:  Biovector Therapeutics, S.A. (Labege Cedex, FR)

Filed:  September 30, 1999

PCT NO:  PCT/FR97/02332

102(e) Date:  September 30, 1999

PCT PUB. Date:  July 9, 1998

Foreign Application Priority Data:  Dec 27, 1996[FR] (96 16121)

The invention relates to an ionic conjugate, which is stable in a biological medium, and which is comprised of a particle vector with at least one cationic, nonliquid, hydrophilic nucleus and of polyanionic oligonucleotides. The invention further concerns the pharmaceutical compositions containing these conjugates and the use of a particle vector to carry the oligonucleotides to the cells.

SUMMARY OF INVENTION

The applicants have developed their expertise in the preparation of synthetic particle vectors, designated by the general term of Supramolecular Biovector nanoparticules (SMBV), or biovectors, which can fix and advantageously target different substances or active ingredients, and are therefore useful for the manufacture of pharmaceutical compositions.

A first type of supramolecular biovector, and the process for its preparation, has been described in European Patent No. 344,040.

These particles include:

a central nonliquid hydrophilic nucleus made up of a polysaccharide or oligosaccharide nucleus, naturally or chemically crosslinked, which can be modified by various ionic groupings;

a layer of fatty acids grafted by covalent bonds to the nucleus; and

one or more lipid layers, made up specifically of phospholipids.

The developments of this first generation of Supramolecular Biovector nanoparticule shave led the applicants to design and prepare new Supramolecular Biovector nanoparticules with improved properties, particularly in terms of the active ingredients which are transported. The international patent applications (PCT) published under the Nos. WO 92/21329, WO 92/21330, WO 94/23701, WO 94/20078 and WO 96/06638, describe these supramolecular biovectors, their manufacture, their combination with various active principles, and their use for the preparation of pharmaceutical compositions.

The international patent applications (PCT) published under the Nos. WO 92/21329, WO 92/21330, WO 94/23701, WO 94/2278 and WO 96/06638 are incorporated herein by reference.

The combination of a Supramolecular Biovector with oligonucleotides has been mentioned in the International Patent Application PCT No. WO 92/21330, in the context of a Supramolecular Biovector having a selective tropism for a type of cell, and therefore presenting the following particular structure:

a nonliquid hydrophilic nucleus

a first layer, lipid in nature, linked to the nucleus by covalent bonds;

a second layer of phospholipids linked to the first layer by hydrophobic interactions; and

molecules of apolipoprotein B grafted onto the layer of phospholipids or protein or peptide ligands, which can specifically recognize the cellular receptors of the LDLs.

The possibility of the combination of a Supramolecular Biovector with oligonucleotides has also been presented by the inventors at the 21^st International Symposium on controlled release of Bioactive Materials, Jun. 27-30, 1994, in Nice (France). At the time of this presentation the proposal was made that the Supramolecular Biovector nanoparticules should be used to increase the therapeutic effectiveness of unmodified oligonucleotides. The Supramolecular Biovector nanoparticules in question were of the type described in International Patent Application No. PCT WO 92/21329, i.e., made up of a nucleus of polysaccharides crosslinked by epichlorhydrin, functionalized by glycidyltrimethylammonium chloride to make positive charges appear, and covered with a first layer of fatty acids and a second lipid layer. It was proposed that the acylated nuclei be combined with the oligonucleotides by simple mixing in an aqueous medium, then creating the external lipid layer by mixing the acetylated nuclei combined with oligonucleotides with a lipid solution.

The research work carried out by the applicants since this symposium has allowed them to demonstrate, as indicated in the examples given below, that the effectiveness of a combination of a Supramolecular Biovector and oligonucleotides did not result from a simple incorporation of the oligonucleotides in the supramolecular biovector, but requires the formation of an ionic conjugate which should be stable in a biological medium presenting the ionic characteristics of the blood plasma, and allow the transport, protection, and addressing of oligonucleotides, antisenses or ribozymes, in the cells and advantageously in the cytoplasm. In addition, it was necessary to prepare conjugates whose level of combination of oligonucleotides is maximal, in order to assure the greatest effectiveness of the desired effect.

Such a stable ionic conjugate established between, on the one hand, the positive charges of the nucleus and, on the other hand, polyanionic oligonucleotides, can be obtained only if the rate of crosslinkage of the matrix and the level of positive charges of the nucleus are rigorously defined.

The studies that have led to the present invention were carried out with the applicants' understanding of the processes of making supramolecular biovectors, which allowed them to study the possibilities of modulation of the degree of crosslinkage of the polysaccharide nucleus and adjustments of the level of cationic charges of the nucleus.

Therefore, the purpose of the invention is to provide new means of transport, protection, and addressing of oligonucleotides in the cells, particularly for therapeutic purposes. This goal is reached with a new ionic conjugate, which is stable in a biological medium, of a particle vector having at least one nonliquid cationic hydrophilic nucleus and polyanionic oligonucleotides, making it possible to transport into the cells and to protect said oligonucleotides, which are capable of hybridizing with a cellular mRNA.

Claim 1 of 30 Claims

What is claimed is:

1. An ionic conjugate, stable in a biological medium, of a particle vector comprising at least one non-liquid cationic hydrophilic nucleus, and polyanionic oligonucleotide, wherein said nucleus comprises a matrix of polysaccharides or oligosaccharides naturall or chemically crosslinked in an amount essentially equal to crosslinkage obtained between 10 and 15 mol % of epichlorhydrin per mole of ose in the polysaccharides or oliposaccharides.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.