Lipids and their use, for example, in liposomes

Details Lipids and their use, for example, in liposomes Lipids and their use, for example, in liposomes

1998-10-14

2002-10-01

Kishore, Gollamudi S. (Department: 1615)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C564S291000

Reexamination Certificate

active

06458381

ABSTRACT:

This invention relates to lipid compounds and their use, for example for the transport of biologically active substances or molecules in cells.
Liposomes are spherical, self-closed structures composed of lipid bilayers which entrap in their interior a portion of the solvent, in which they float. They may consist of one or more concentric membranes, and their size ranges from several nanometers to several dozens of micrometers.
Liposomes are mostly made from amphiphilic molecules which can be characterized by having a hydrophilic (often named the polar head) and a hydrophobic group (nonpolar tail) on the same molecule. In most cases, liposome-forming molecules are not soluble in water. However, under certain circumstances, they form colloidal dispersions.
Liposomes can be large or small and may be composed from one to several hundred of concentric bilayers. With respect to the size and the nature of the layer (lamellae), they can be classified as multi-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar vesicles LUVs).
SUVs have a diameter from 20 to 600 nm and consist of a single lipid bilayer which surrounds the interior aqueous compartment. LUVs have a diameter from 600 to 30000 nm. MLVs vary greatly in size for up to 10000 nm and contain more than one lipid bilayer, therefore they are multi-compartmental in their structure.
Liposomes can be produced in a number of ways. The so-called “thin-film hydration” method results in the formation of heterogeneous dispersions of predominantly MLVs. By using charged lipid compositions rather high fractions of LUVs can be produced. Said dispersions can be further treated (mechanically, electrostatically or chemically) in order to produce solutions of SUVs. Most frequently these methods include extrusion through filters with pores of different diameter, or sonication.
Alternatively, liposomes can be prepared by lyophilization, where the lipid-film is then dissolved in a volatile solvent (for example tert-butyl alcohol), frozen and lyophilized.
A variety of methods for preparing liposomes have been described in the periodical and patent literature: Szoka and Papahadjopoulos in: Ann. Rev. Biophys. Bioeng. 9, 467-508 (1980) as well as U.S. Pat. Nos. 4,229,360, 4,241,046, 4,235,871.
The most important liposome feature is their ability to dissolve, protect and carry hydrophilic or hydrophobic molecules. For negatively charged drugs, including some proteins, positively charged liposomes can be used. Improvements in therapy were observed, despite the known fact that positively charged liposomes can be toxic.
Various DNA transfection methods have been developed in the past twenty years. These methods include the calcium phosphate precipitation method, DEAE-dextran method, electroporation method, microinjection, receptor mediated endocytosis, liposomes and viral vectors. However, most of these methods posess some significant drawbacks: they are either too inefficient, or too toxic, or too complicated and tedious to be effectively adapted to biological and therapeutical protocols both in vitro and in vivo. For instance, the most frequently used in vitro calcium phosphate precipitation method is too inefficient (average transfection frequency of 1 in 10
4
cells). Electroporation is much more efficient than the calcium phosphate method. However, this method is too aggressive (maximum efficiency is obtained at about 50% of cell death) and, in addition, this method requires a special apparatus. Microinjection is efficient, but it is too tedious and not practical. All these methods cannot be used in vivo.
A receptor mediated endocytosis method involves polylysine as a basic polymer for interacting and packaging of DNA. Polylysine has been modified with different ligands (transferrin, insulin, asialoorosomukoid, or galactose) in order to target modified protein-DNA complexes to cell surface receptors: Wu, G. Y. et al. in: J. Biol. Chem. (1987) 262:4429-4432, Cotten, M. et al. in: Proc. Natl. Acad. Sci. (USA) (1990) 87:4033-4037, Huckett, B. et al. in: Biochem. Pharmacol. (1990) 40:253-263, Plank, C. et al. in: Bioconjugate Chem. (1992), 533-539. The method has been dramatically improved by use of inactivated adenovirus to facilitate exit of DNA from endosomes, see: Wagner, E. et al. in: Proc. Natl. Acad. Sci. (USA) (1992) 89:6099-6103, Christiano, R. J. et al. in: Proc. Natl. Acad. Sci. (USA) (1993) 90:2122-2126. The major disadvantage of the described approach includes an inherent inability to control the protein conjugation chemistry and to prepare such conjugates in a reproducible fashion.
At present the best transfection efficiencies both in vitro and in vivo are obtained with retrovirus, adenovirus, and some others, see for example: Kerr, W. G. and Mule, J. J. in: J. Leucocyte Biol. (1994) 56:210-214, Hwu, P. and Rosenberg, S. A. in: Cancer Detect. Prevent. (1994) 18:43-50, Rosenfeld, M. A. et al. in: Cell (1992) 68:143-155. Nevertheless, the use of viral vectors poses several considerations including the requirement of extensive cell culture manipulations, low titers for certain virus systems and the cell tropism of the virus. In addition, immune reactivity against viral vectors may cause problems. Most importantly, the safety issues related to the use of viral vectors are not completely resolved to date.
Liposomes have also been used to introduce DNA into cells both in vitro and in vivo. The most successful liposome systems use different cationic lipids like dioleyloxypropyl-trimethylammonium (DOTMA, which forms a reagent in combination with phosphatidylethanolamine (PE)), dioleoyloxypropyl-trimethylammoniummethyl sulfate (DOTAP), dimethylaminoethane-carbamoyl cholesterol, dioctadecylamidoglycylspermine, 2,3-dioleyloxy-N-(2(sperminecarboxamido)ethyl)-N,N-dimethyl-1 propanamine (DOSPA), which in combination with PE forms a reagent, see: Felgner, P. L. in: Proc. Natl. Acad. Sci. (USA) (1987) 84:7413-7417, U.S. Pat. No. 5,208,036, Leventis, R. and Silvius, J. R. in: Biochimica et Biophysica Acta (1990), 124-132, Gao, X. and Huang, L. in: Biochem. Biophys. Res. Commun. (1991) 179:280-285, Behr, J.-P. et al. in: Proc. Natl. Acad. Sci. (USA) (1989) 86:6982-6986.
The advantage of using the above mentioned compounds is that the cationic liposome is simply mixed with DNA and added to the cell. Transfection efficiency is usually high when compared to other physical methods of DNA transfer. Besides for delivery of DNA, a specific compound has been used to deliver mRNA and proteins into cultured cells, see: Malone, R. et al. in: Proc. Natl. Acad. Sci. (USA) (1989) 86:6077-6081, and Debs, R. et al. in: J. Biol. Chem. (1990) 265, 10189-10193. Some of the above mentioned compounds have been used to transfect reporter or therapeutically utile genes in vivo, see: Nabel, G. J. et al. in: Proc. Natl. Acad. Sci. (USA) (1993) 90:11307-11311, Zhu, N. et al. in: Science (1993) 261:209-211. Finally, a DNA transfection protocol has been developed that makes use of the cyclic cationic peptide gramicidin S and PE, see: Legendre, J.-Y. and Szoka, F. C. in: Proc. Natl. Acad. Sci. (USA) (1993), 90:893-897. The above mentioned system takes advantage of the DNA binding ability and the membrane destabilization properties of gramicidin S.
The main disadvantage of cationic liposomes includes their relatively high cytotoxicity. In addition, most of the above mentioned compounds are not active or show highly reduced activity in the presence of serum. Most of them need the use of PE, possibly because PE can form intramembrane lipid intermediates which facilitate membrane fusion. Studies on the mechanism responsible for transfection using the cationic lipids have not been fully addressed to date. The need exists, therefore, for a less toxic, non-infectious and more efficient delivery of biological molecules into the cytoplasm and nuclei of living cells.
The object of the present invention is to overcome the above mentioned and other drawbacks of the state of the art. According to a first aspect there are lipid compounds to be provided to allow an impr

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