Lipopolyamines, and the preparation and use thereof

Details Lipopolyamines, and the preparation and use thereof Lipopolyamines, and the preparation and use thereof

2000-03-29

2001-08-28

Carr, Deborah D. (Department: 1621)

Organic compounds -- part of the class 532-570 series

Organic compounds

Fatty compounds having an acid moiety which contains the...

C554S104000, C564S192000, C564S193000, C564S403000, C564S511000, C564S512000, C514S625000, C514S626000, C424S450000

Reexamination Certificate

active

06281371

ABSTRACT:

This application is a 371 of PCT/EP98/05156 filed Aug. 13, 1998.
Positively charged lipids (J. P. Behr, Bioconjugates Chem. 5,382-389, 1994) are used in the form of liposomes, micelles or per se, for the introduction of biologically active substances, such as peptides, peptoids, proteins, PNA and antiviral active substances, but especially DNA, RNA, antisense DNA/RNA or ribozymes, into eukaryotic cells (for example mammalian, plant or insect cells). Lipopolyamines are a special class of cationic lipids that exhibit comparatively outstanding transfection properties. “Transfection” is to be understood as meaning the introduction of hereditary material into eukaryotic cells.
The need to introduce DNA (for example plasmids, cosmids, single-stranded or double-stranded), RNA or related classes of substances, such as antisense DNA/RNA or ribozymes, into eukaryotic cells in order to be able, for example, to carry out gene therapy successfully, has led to the development of numerous transfection methods. A large number of methods are known for the introduction of nucleic acids into eukaryotic cells, especially mammalian cells, such as, for example, the CaPO
4
precipitation method, the DEAE/dextran method, synthetic polyamines (polyethyleneimine, polylysine, PAMAM dendrimers), methods that use receptor-mediated endocytosis, electroporation, microbombardment, microinjection and methods that use viral capsids as DNA carriers. A further method is termed “lipofection” (P. L. Felgner et al., Proc. Natl. Acad. Sci. USA 74, 7413 1987) which makes use of the fact that synthetic cationic lipids, in the form of liposomes, micelles or per se, form complexes with negatively charged DNA. By so adjusting the relative amounts of DNA and cationic lipid to produce complexes having a net positive charge, the complexes obtained have a high affinity for the negatively charged membrane surface of eukaryotic cells. When such DNA/lipid complexes contact cells, the introduction of the genetic material into the cell results. The exact mechanism by which the DNA gets into the cells is still largely unknown, but it is assumed that either fusion of the cationic lipids with the anionic cell membrane occurs, with simultaneous delivery of the DNA into the interior of the cell, or that the DNA/lipid complexes pass in their entirety into the cell by means of a natural transport mechanism of the cells, so-called endocytosis, and the DNA is then released.
Liposomes are, as a rule, spherical arrangements of lipids in aqueous solutions having a “bilayer structure” and are typically divided into three classifications (see N.Y. Academy Sciences Meeting: “Liposomes and their use in Biology and Medicine”, December 1977): multilamellar vesicles (MLV, up to 10,000 nm), small unilamellar vesicles (SUV, 20-50 nm) and large unilamellar vesicles (LUV, 600-30,000 nm). A number of methods for the production of liposomes is known and these are described in “Liposome Technology” (Gregoriadis, CFC Press, New York 1984), in “Liposomes” (Ostro, Marcel Dekker, New York 1987) or in review articles by
Lichtenberg et al. (Methods Biochem. Anal. 33, 337-462, 1988), Pagano and Weinstein (Ann. Rev. Biophysic. Bioeng. 7, 435-468, 1978) or Szoka and Papahadjopoulos (Ann. Rev. Biophysic. Bioeng. 9, 467-508, 1980). Known methods include, for example, the “reverse-phase evaporation” method and the extrusion method, in which a lipid solution is pressed through a microporous membrane.
Liposomes are typically also prepared in the following manner: the lipids are taken up in an organic solvent By vaporisation of the solvent under a stream of nitrogen, a thin film of lipid is produced on the wall of the glass vessel. The addition of water or aqueous buffer solution hydrates that film. The solution obtained is finally treated with ultrasound.
Cationic lipids are becoming increasingly important in gene therapy. In such therapy, body cells are transfected in vivo by various methods, by administering complexes of carrier and DNA intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, intranasally, into fluid spaces or directly into tumours, or by removing, transfecting and reimplanting body cells. Until some time ago, a favoured method was the introduction of the genetic material by viral carrier, but that method carries the risk of retromutation to a pathogenic virus. Furthermore, the DNA introduced is stably incorporated into the genotype, so it is not possible to control the therapy or to return the cells to their original state. In addition, viral carriers have restrictions regarding the size of the DNA to be introduced. Modified DNA or RNA is not transferred by viruses. Also, only dividing cells can be transfected by that method.
Other risks to be considered when using viral carrier systems are the possible activation of oncogenes and an immune reaction of the treated organism.
Transfection with cationic lipids, on the other hand, is not subject to those restrictions. The transfection is usually transient, that is to say, the transfected DNA or RNA is expressed only for a certain period, since it is not incorporated in the genotype and over time is degraded by nucleases. In that way gene therapy can be measured and made reversible. There are no restrictions in terms of the size of the DNA and, in addition, it is also possible by means of cationic lipids to introduce modified DNA or RNA (for example antisense DNA/RNA, or ribozymes stabilised by the incorporation of modified nucleotides) into cells. Also, non-dividing cells, such as, for example, nerve cells, can be transfected by cationic lipids.
Furthermore, cationic lipids have not so far been found to have any immunogenic behaviour in in vivo tests.
While the in vivo use of microinjection and electroporation does not appear possible for process-related reasons, the CaPO
4
and DEAE/dextran methods exhibit poorer transfection efficiency compared with lipofection.
The cationic lipids include a class of lipids, so-called lipopolyamines, that utilizes the known high affinity between polyamines (for example spermine, spermidine) and DNA for transfection. The polyamines in that class of lipids have a linear or branched structure and contain ethylene, propylene or butylene groups between the amino functions. The polyamines are bound in a wide variety of manners to a lipophilic radical.
For example, sperrnine, which is positively charged at physiological pH, is linked to a hydrophilic radical in some cases by way of a spacer. Spermine forms stable complexes with DNA and similar compounds by being bound in the groove of the DNA by hydrogen bridge bonding. The first such lipospermnine derivatives were synthesised by Behr, J. P. et al. (Proc. Natl. Acad. Sci. USA 86; 6982-6986; 1989; EP 0 394 111). In that process carboxyspermine was linked by way of a spacer to two different hydrophilic radicals. The structure of the resulting 5-carboxyspermylglycinedioctadecylamide (DOGS) is as follows:
DOGS is available commercially as Transfectam™ (Promega). The second compound developed by Behr et al. is dipalmitoylphosphatidylethanolamine-5-carboxyspermylamide (DPPES):
A further lipospermine derivative is claimed by P. L. Felgner et al. in WO 91/16024 under the name L-spermine-5-carboxy-3-(DL-1,2-dioleoyldimethylaminopropyl-&bgr;-hydroxyethylamine). In the Patent Specification, however, L-spermine-5-carboxyl-3-(DL-1,2-dipalmitoyldimethylaminopropyl-&bgr;-hydroxyethylamine) is described:
In WO 94/05624, Gebeyehu, G. et al. describe the compound N-[N-(5-carboxyspermyl)aminoethyl]-N,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propaneammonium tetra(trifluoroacetate), which is available commercially as Lipofectamin™ (Gibco-BRL: Life Technologies Inc.):
In WO 97/00241, von Der Eltz et al. describe the compound 2-(6-carboxyspermyl)-1,3-dioleoyloxypropylamide, which has been marketed under the name DOSPER (Boehringer Mannheim GmbH):
The compounds mentioned so far were all prepared by amide or ester linkage of a linear polyamine carrying a carboxy function as side chain, i.e. by linkage of carbox

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