Use of a cationic amphipathic compound as a transfection agent,

Details Use of a cationic amphipathic compound as a transfection agent, Use of a cationic amphipathic compound as a transfection agent,

1997-03-03

2000-09-26

Geist, Gary

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

514529, 560250, 560251, 560252, 560253, 560261, 560262, 560263, 560264, 560265, 560266, 424812, 4353201, A61K 31215, A61K 3900

Patent

active

061242705

DESCRIPTION:

BRIEF SUMMARY
The invention relates to the field of cationic amphipathic compounds and to their use in particular as transfection agent or as vaccine additive.
Transfection, that is to say the introduction, without damage, into a living eukaryotic cell of DNA or mRNA (messenger RNA) capable of being expressed, constitutes an alternative approach to the intracellular introduction of polypeptides and proteins. This technique finds application in numerous fields ranging from cell biology to therapy. In cell biology, transfection techniques can be used especially to study the intracellular role of cloned gene products and to study the regulation of gene expression. Transfection is also used in gene therapy for the correction of genetic disorders; it also finds application in the field of therapeutic peptides and in immunization with the development of polynucleotide vaccines.
There are known in the prior art numerous transfection methods, of which in particular precipitation of DNA and calcium phosphate or DEAE-dextran, or alternatively the use of cationic lipids in the form of liposomes which form complexes with negatively charged polynucleotides and facilitate their transmembrane passage. Among the cationic lipids known, there may be mentioned: marketed in combination with a neutral lipid, DOPE (or dioleoylphosphatidyl-ethanolamine) in the form of liposomes, by the company GIBCO BRL under the name Lipofectin.TM., supplied by the company SEPRACOR under the name Transfectam.TM., and DOSPA (2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-propaneammon ium trifluoroacetate) supplied by the company GIBCO BRL under the name Lipofectamine.TM., (dimethyldioctadecylammonium bromide) marketed in combination with a neutral lipid in the form of liposomes by the company GIBCO BRL under the name Transfectace.TM., (3.beta.-[N-(N'N'-dimethylaminoethane)carbamoyl]-cholesterol) (Cf. X. Gao and L. Huang, Biochem. Biophys. Res. Commun., 1979, 280-285, 1991), DOTAP(1,2-dioleoyloxy-3-(trimethylammonio)propane) supplied by the company BOEHRINGER MANNHEIM.
Although a number of these products are commercially available and are effective for the transfection of cells in culture, their efficacy in vivo is limited. The need therefore exists for new transfection agents capable of promoting the passage of polynucleotides across the negatively charged cell membrane, which are not or not very toxic and which are effective in vivo.
The aim of the invention is to provide such transfection agents having fusogenic properties with the cell membrane or the endosome membrane.
To achieve this aim, the subject of the invention is a cationic amphipathic compound of formula I: ##STR2## in which: A represents a single bond, an NH--R' group, or an ##STR3## group, where --R'-- is an aliphatic chain containing from 1 to 22 carbon atoms, which is linear or branched, optionally substituted, saturated or unsaturated, optionally interrupted by one or more heteroatoms O, S, N, as well as by one or more aromatic or saturated or unsaturated heterocyclic or carbocyclic radicals, higher alkyl or acyl group, alkylene radical --(CH.sub.2).sub.n -- where 1.ltoreq.n.ltoreq.6, atoms, which is optionally substituted, optionally interrupted by one or more heteroatoms chosen from O, S, N or by one or more aromatic or saturated or unsaturated heterocyclic or carbocyclic radicals, form together with the nitrogen atom to which they are linked a quinuclidino, piperidino, pyrrolidino or morpholino group,
According to one specific characteristic of the invention, R.sub.7, R.sub.8 and R.sub.9 are all identical and represent the methylene group --CH.sub.2 --.
According to another characteristic of the invention, R.sub.4, R.sub.5 and R.sub.6 are all identical and represent the methyl group CH.sub.3 --.
According to another characteristic, R.sub.1, R.sub.2 and R.sub.3 are all identical and represent a higher acyl group such as dodecanoyl or hexadecanoyl.
According to another characteristic, A represents a group ##STR4## where 1.ltoreq.m.ltoreq.22,
According to a specific mode of the

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Kunitake et al., "Bilayer Membranes . . . Amphipiles", J. Am. Chem. Soc., vol. 106, pp. 1978-1983, 1984.
Felgner et al., "Enhanced Gene . . . Formulations", J. of Biol. Chem., vol. 269, No. 4, pp. 2550-2561, Jan. 1994.
T. Kunitake et al., "Bilayer Membranes of Triple-Chain Ammonium Amphiphiles" J.Am.Chem.Soc. (1984) 106:1978-1983.

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