Glycerolipid compounds useful for the transfer of an active...

Details Glycerolipid compounds useful for the transfer of an active... Glycerolipid compounds useful for the transfer of an active...

2001-03-02

2004-06-01

McGarry, Sean (Department: 1635)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C536S023100, C424S001110, C424S009100, C424S009341, C424S450000, C435S006120, C435S320100, C435S325000, C435S375000, C560S224000, C530S323000

Reexamination Certificate

active

06743781

ABSTRACT:

The present invention relates to new glycerolipid compounds and new compositions containing them. More particularly, the present invention relates to the use of said compounds or of said compositions to prepare a vector for transferring an active substance, in particular a therapeutically active substance comprising negative charges, in particular a polynucleotide, into a target cell, particularly a vertebrate cell, and more particularly a mammalian cell.
The transfer of a gene into a given cell is the very basis of gene therapy. This new technology, whose field of application is vast, makes it possible to envisage the treatment of serious diseases for which the conventional therapeutic alternatives are not very effective, or are even inexistent, and applies to diseases which are either of genetic origin (hemophilia, cystic fibrosis, myopathy and the like) or acquired (cancer, AIDS and the like).
During the past 30 years, numerous tools have been developed which allow the introduction of various heterologous genes into cells, in particular mammalian cells. These different techniques may be divided into two categories. The first category relates to physical techniques such as microinjection, electroporation or particle bombardment which, although effective, are greatly limited to applications in vitro and whose implementation is cumbersome and delicate. The second category involves techniques relating to molecular and cell biology in which the gene to be transferred is combined with a vector of a biological or synthetic nature which promotes the introduction of said material.
Currently, the most effective vectors are viral, in particular adenoviral or retroviral, vectors. The techniques developed are based on the natural properties which these viruses have to cross the cell membranes, to escape degradation of their genetic material and to cause their genome to penetrate into the nucleus. These viruses have already been the subject of numerous studies and some of them are already used experimentally as vectors for genes in humans for the purpose, for example, of a vaccination, an immunotherapy or a therapy intended to make up for a genetic deficiency. However, this viral approach has many limitations, in particular because of the limited capacity for cloning into the viral genome, the risks of spreading in the host organism and in the environment the infectious viral particles produced, the risk of artefactual mutagenesis by insertion into the host cell in the case of retroviral vectors, and the high induction of immune and inflammatory responses in vivo during the therapeutic treatment, considerably limiting the number of administrations which can be envisaged (McCoy et al., 1995, Human Gene Therapy, 6, 1553-1560; Yang et al., 1996, Immunity, 1, 433-442). These numerous disadvantages, in particular in the context of a use in humans, have led several teams to develop alternative systems of transferring polynucleotides.
Several non-viral methods are currently available. By way of example, there may be mentioned coprecipitation with calcium phosphate, the use of receptors mimicking viral systems (for a review see Cotten and Wagner, 1993, Current Opinion in Biotechnology, 4, 705-710), or the use of polymers such as polyamidoamine (Haensler and Szoka, 1993, Bioconjugate Chem., 4, 372-379) or of polymer such as those presented in WO 95/24221 describing the use of dendritic polymers, the document WO 96/02655 describing the use of polyethyleneimine, or of polypropyleneimine and the documents U.S. Pat. No. 5,595,897 and FR 2,719,316 describing the use of conjugates of polylysine. Other non-viral techniques are based on the use of liposomes whose value as agent allowing the introduction, into cells, of certain biological macromolecules, such as for example DNA, RNA, proteins or certain pharmaceutically active substances, has been widely described in the literature. To this end, several teams have already proposed the use of cationic lipids which have a high affinity for the cell membranes and/or the nucleic acids. Indeed, although it has been shown, in the case of nucleic acids, that this type of macromolecule is capable of crossing the plasma membrane of some cells in vivo (WO 90/11092), it is nevertheless the case that the observed transfection efficiency is still highly limited, because of in particular the polyanionic nature of the nucleic acids which prevent their passage across the cell membrane, which itself has a negative net apparent charge. Since 1989 (Felgner et al., Nature, 337, 387-388), cationic lipids have been presented as molecules which are advantageous for promoting the introduction of large anionic molecules, such as nucleic acids, into certain cells. These cationic lipids are capable of complexing anionic molecules, thus tending to neutralize the negative charges on said molecules and to promote their coming close to the cells. Many teams have already developed various cationic lipids. By way of example, there may be mentioned DOTMA (Felgner et al., 1987, PNAS, 84, 7413-7417), DOGS or Transfectam™ (Behr et al., 1989, PNAS, 86, 6982-6986), DMRIE and DORIE (Felgner et al., 1993, Methods 5, 67-75), DC-CHOL (Gao et Huang, 1991, BBRC, 179, 280-285), DOTAP™ (McLachlan et al., 1995, Gene Therapy, 2,674-622) or Lipofectamine™, as well as those described in Patent Applications WO9116024 or WO9514651.
More particularly, Application WO9405624 describes cationic lipids of formula:
in which the R radicals are in particular octadecenyl radicals, the Z radicals are C
1
-C
13
alkyl or —C(═O)—(C
1
-C
13
) alkyl or acyl radicals, q is an integer from 1 to 6, X is in particular a short polyamine chain, such as spermine, spermidine, carboxyspermine or polylysine.
Applications EP 685457 and EP 685234 describe in particular cationic compounds of formula:
in which R is in particular a hydrocarbon chain having 10 to 30 carbon atoms, saturated or otherwise, A may in particular be chosen from the groups —O—(C═O)— and —NH—(C═O)—, n varies from 0 to 4 and R
1
is an alkyl or a short aminoalkyl chain in which the primary amine is substituted with an alkyl having 2 to 8 carbon atoms. These compounds have a low hemolytic activity and make it possible in vitro to introduce into HelaS3 cells a double-stranded RNA capable of acting as growth inhibiting agent.
Application DE 19521412 describes compounds which also comprise a nonpolar part and a polar part of formula:
in which p varies from 1 to 6, q varies from 0 to 2, R is either C(═O)—C1-23 or C1-23, saturated or otherwise, and Z is a peptide, an aminoacid or a branched amino structure. These cationic compounds allow the in vitro transfection of cells in culture.
However, several studies (by way of example, see Mahato et al., J. Pharm. Sci., 1995, 84, 1267-1271, Thierry et al., 1995, P.N.A.S., 92, 9742-9746) have demonstrated that the efficiency of transferring the anionic macromolecule into cells could vary depending in particular on the interaction between the complexes and the cell membranes, the cell considered, the lipid composition of the cationic compounds, the size of the complexes formed with the anionic molecules and more particularly the ratio between the positive and negative charges on the different components of said complex. The mechanisms which allow in particular the interaction of the complexes with the cell membranes and the transfer of the complexes into the cell are still to a large extent poorly understood and researchers proceed in their studies based on a highly empirical approach. It is consequently desirable to provide other cationic lipids possibly having improved properties or properties which are different from the cationic lipids already described.
The Applicant has now identified new glycerolipid compounds, which can be provided in cationic form, useful in particular for transferring an active substance comprising negative charges, in particular a polynucleotide, into a target cell, whose use may be envisaged in particular in vivo in the context of a gene therapy.
Accordingly, the subject of th

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