Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-02-12
2001-09-18
Priebe, Scott D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S04400A, C514S252120, C544S400000, C424S450000, C435S455000, C435S458000, C530S403000, C530S405000
Reexamination Certificate
active
06291423
ABSTRACT:
The present invention relates to novel complexes comprising at least one lipid compound as defined below and at least one therapeutically active substance. More particularly, the present invention relates to the use of said complexes for transferring at least one therapeutically active substance containing negative charges, in particular a polynucleotide, into a target cell, particularly a vertebrate cell, and more particularly a mammalian cell.
Gene transfer into a given cell is the very basis of gene therapy. This technology, whose field of application is vast, makes it possible to envisage treating serious diseases for which the standard alternative therapies are ineffective, or even nonexistent, and relates equally to diseases of genetic origin (hemophilia, mucoviscidosis, myopathy, etc.) and acquired diseases (cancer, AIDS, etc.).
In the course of the last 30 years, many tools have been developed for introducing various heterologous genes into cells, in particular mammalian cells. These various techniques can be divided into two categories. The first category relates to physical techniques such as microinjection, electroporation or particle bombardment, which, although effective, are largely limited to in vitro applications and are cumbersome and difficult to carry out. The second category involves techniques relating to molecular biology and cell biology, for which the gene to be transferred is combined with a vector of biological or synthetic nature which promotes the introduction of said material.
The vectors which are currently the most effective are viral vectors, in particular adenoviral or retroviral vectors. The techniques developed are based on the natural properties which these viruses have for crossing cell membranes, avoiding the degradation of their genetic material and enabling their genome to penetrate into the nucleus. These viruses have already been the subject of many studies and some of them are already used experimentally as gene vectors in man for the purpose, for example, of a vaccination, an immunotherapy or a therapy aimed at compensating for a genetic deficiency. However, this viral approach has many limitations, in particular on account of the restricted cloning capacity in the viral genome, risks of dissemination of the infectious viral particles produced into the host body and into the environment, risk of artefactual mutagenesis by insertion into the host cell in the case of retroviral vectors, and strong induction of immune and inflammatory responses in vivo during therapeutic treatment, which considerably limits the number of administrations which can be envisaged (Mc Coy et al, 1995, Human Gene Therapy, 6, 1553-1560; Yang et al., 1996, Immunity, 1, 433-442). These many drawbacks, in particular in the context of a use in man, have led several teams to develop alternative systems for transferring polynucleotides.
Several non-viral methods are available at the present time. Mention is made, for example, of the coprecipitation with calcium phosphate, the use of receptors which mimic viral systems (for a revue 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 a polymer such as those given in WO 95/24221 which describes the use of dendritic polymers, document WO 96/02655 which describes the use of polyethyleneimine or of polypropyleneimine and documents U.S. Pat. No. 5,595,897 and FR 2,719,316 which describe the use of polylysine conjugates. Other non-viral techniques are based on the use of liposomes whose value as agents for introducing into cells pharmaceutically active substances such as, for example, DNA, RNA or proteins has been widely described in the literature. To this end, several teams have already proposed the use of cationic lipids which have a strong affinity for cell membranes and/or nucleic acids. The reason for this is that, although it has been shown, in the case of nucleic acids, that this type of macromolecule is capable of crossing the plasma membrane of certain cells in vivo (WO 90/11092), the fact nevertheless remains that the efficacy of the transfection observed is still very limited, in particular on account of the polyanionic nature of nucleic acids which prevents them from crossing the cell membrane, which itself has a net negative apparent charge. Since 1989 (Felgner et al., Nature, 337, 387-388) cationic lipids have been presented as advantageous molecules for promoting the introduction of large anionic molecules, such as nucleic acids, into certain cells. These cationic lipids are capable of complexing with anionic molecules, thus tending to neutralize the negative charges on said molecules and to promote their approach toward cell walls. Many teams have already developed various cationic lipids. As examples, mention may be made of 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 and 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 WO 91/16024 or WO 95/14651 or WO-A-94/05624.
More particularly, patent application WO-A-95/14651 describes cationic lipids of formula:
in which R is a linear chain, an aliphatic alkyl group of 5 to 29 carbon atoms, Y is —CH
2
— or CO, R′ is a lower alkyl radical, m is an integer from 0 to 7, n is 0 or 1, the total number of carbon atoms in R and (CH
2
)
m
being at least equal to 10.
However, several studies (as examples, 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 efficacy of the transfer into cells of the anionic macromolecule can vary as a function in particular of 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 of positive and negative charges on the different components of said complex. The mechanisms which in particular allow the interaction of the complexes with cell membranes and the transfer of the complexes into the cell are still largely unknown and researchers are proceeding in their studies with a highly empirical approach. Other factors such as, for example, the formation of complexes, the stability, the behavior in vivo, or optionally their toxicity, also make the choice of lipids not obvious in principle. It is consequently desirable to propose other cationic lipids, which make it possible to design novel non-viral vectors or lipid complexes optionally having properties that are better than or different from those already described.
The present invention relates, firstly, to a complex comprising at least one lipid and at least one therapeutically active substance, in particular containing negative charges, which can be used for transferring said substance into a target cell, characterized in that said lipid is of formula I:
in which:
n
1
and n
2
, which may be identical or different, are integers chosen from 0 and 1,
R
1
and R
2
, which may be identical or different, are:
a) chosen from the group consisting of a hydrogen atom and alkyl radicals of 1 to 6 carbon atoms which are optionally substituted, independently of each other, with a hydroxyl radical, or
b) according to a specific case for which n1=n2=1, R1 and R2 can together form a divalent alkylene chain of 2 to 3 carbon atoms (C2-C3),
R
3
and R
4
, which maybe identical or different, are alkyl radicals of 1 to 6 carbon atoms or can together form a divalent alkylene chain of 2 to 3 carbon atoms (C2-C3), m and p, which may be identical or different, are integers between 1 and 10,
R
5
and R
6
, which may be identical or different, are chosen from the group consisting of the radicals of formula:
1) R
7
C(═O)—X— in which:
X=NH, O or S,
R
7
is a l
Bischoff Rainer
Heissler Danis
Nazih Abdesslame
Brunovskis Peter
Burns Doane Swecker & Mathis L.L.P.
Priebe Scott D.
Transgene S.A.
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