United States Patent:   5,980,935

Appl. No.:  648558

The present invention relates generally to a non-toxic lipid conjugated with a cationic amino acid containing a guanidino group. Specifically, the naturally-occuring lipid DOPE is combined with the naturally-occurring amino acid Arginine. These compounds are useful for encapsulating and delivering pharmaceuticals and poly and oligonucleotides. These compound improve over current compounds, because they are composed of non-toxic and, in the case of Arg-DOPE, natural components, and therefore result in minimal unwanted side effects. Methods of use of the cationic lipids are also claimed.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates, inter alia, to materials used in facilitating the delivery of nucleic acids and oligonucleotides into living cells. The utility of such delivery is recognized in the practice of biomedical research and industry, biotechnology, and medicine.

Specifically, the use of cationic lipids for facilitating the entry of functional nucleic acids and oligo nucleotides into living cells has been described in the scientific and patent literature. The array of molecular structures of such lipids, as reviewed, for example, in Remy, cited above, and Behr, cited above, demonstrates that cationic properties of such lipids have been provided by introduction of a positively-charged group, or groups, based on the ammonium function. However, ammonium group (pK 9.2) is a weaker base than guanidine group (pK 12.7) present in the natural protein amino acid arginine, while the use of strong bases such as quaternary ammonium groups renders a molecule of cationic lipid more toxic and less biodegradable by the cell. It is also noteworthy that protamines, natural polypeptides with the highest DNA-compacting ability, have about 60% arginine content.

An underlying concept of the present invention is to employ a guanidine-bearing group with an arginine residue in an amphipatic construct as a candidate for a nucleic acid cellular delivery vehicle which would be readily degraded by cellular enzymes, and fragments resulting from such degradation would be natural ubiquitous metabolites of a cell. In one aspect of the invention, the hydrophilic arm has charged groups, (either negative or zwitterionic in nature), which are useful for forming bilayers in the physiological pH and ionic environment. Liposomal delivery is therefore more advantagous using the present invention. Most specifically, a compound which bears a phosphatidyl group is disclosed in the present invention. For example, N-L-Arginyl-phosphatidyl-ethanolamine is provided by the present invention.

In a practical embodiment of the above-described inventive concept, we have conjugated arginine to a natural phospholipid, phosphatidylethanolamine (PE), by forming an amide linkage between the amino group of PE and carboxy group of arginine. The resulting molecule, N-arginyl-PE (Arg-PE) has the following structure: 

This compound possesses a net cationic charge due to the presence of one acidic and two basic groups, one of the latter being a guanidine group of the arginyl residue. This molecule would be easily split by cellular peptidases into its original components, arginine and PE, both of which are natural cellular constituents.

We have tested Arg-PE for its ability to deliver functional plasmid DNA and phosphorothioate oligonucleotide (PS ON) into living cells. We have also compared this material with an array of comparable materials available commercially. The test showed higher efficiency of the cellular delivery of DNA and PS ON by the invented lipid. It was our unexpected finding that the invented lipid, being essentially a monocationic lipid (bearing the positive net molecular charge equal to one) had the efficiency of DNA delivery superior to that of a polycationic lipid, DOGS, which was reported to have better DNA delivery properties than any of the known monocationic lipids. (Remy and Behr, each cited above.) The inventive lipid was highly active for the DNA delivery into the cells even without the use of a "helper lipid". Helper lipids are required for the activity of previously known monocationic lipids.

Toxicity of most currently available cationic lipids is a limiting factor in their practical uses. We have compared the toxicity of the invented lipid and of the array of commercially available cationic lipids, alone or in combination with DNA for the cultured human cells. This study showed that the invented monocationic lipid Arg-PE has no detectable toxicity in the studied range of concentrations efficient for gene delivery, the property found, again, only in its polycationic counterpart, DOGS, and not generally expected in a monocationic lipid.

The details of the above experiments and others are given in the Examples below.

Therefore, we have prepared the invented material and demonstrated that it meets the purposes of the invention, also showing some unexpected, useful properties that made it superior to the currently available constructs.

It is recognized that the present invention is not limited to the above-described embodiment, Arg-PE, which is merely an example of possible embodiments. More broadly, the invention covers a group of materials whose molecules are capable of bearing a net cationic charge in an aqueous solution and are capable of being degraded in the living cells into non-toxic, metabolizable fragments comprising (1) a guanidino domain as a bearer of the cationic charge; (2) a hydrophobic domain capable of causing the molecule to form micellular structures in aqueous medium and (3) a hydrophilic arm linking together the above two domains.

The delivery of poly-and oligonucleotides into living cells by the invented material uses the same procedure as described in the prior art. Specifically, the invented lipids may be formulated alone, or in the mixture with other (non-cationic) lipids, or even combined with other cationic lipids, in the form of micellular solution, or bilayer vesicles (liposomes), in an aqueous medium, and brought into contact with a polynucleotide (DNA or RNA), or oligonucleotide, prior to administration to the cells. Alternatively, the lipid may be formulated as a solution in a water-miscible organic solvent, such as ethanol, and combined with the poly or oligonucleotide in an aqueous medium prior to administration to the cells.

The invented materials alone are capable of forming bilayer vesicles (liposomes) in an aqueous buffer. Since cationic liposomes are known to be the instruments for intracellular delivery of substances other than nucleic acids (Debs et.al., 265 J. Biol. Chem. 10189 (1990)), the liposomes formed by the invented lipids have utility for the cellular delivery of substances other than poly-or oligonucleotides, such as, for example, proteins and various pharmaceuticals. The present invention therefore provides methods for treating various disease states, so long as the treatment involves transfer of material into cells. In particular, treating the following disease states using the present invention is included within the scope of this invention: cancer, infectious diseases, inflammatory diseases and genetic hereditary diseases.

Claim 1 of 16 Claims

1. A micellar or liposomal composition for enhancing delivery of nucleic acids into cells comprising: at least one nucleic acid, a first moiety and optionally a second moiety,

wherein said first moiety comprises (a) a guanidino domain of the amino acid arginine; (b) a hydrophobic domain capable of causing the molecule to form micellar or liposomal structures in aqueous medium; and (c) a linker joining the guanidino domain and the hydrophobic domain, said linker being a zwitterion in aqueous medium at neutral pH; and

wherein said second moiety is selected from the group consisting of DOPE and cholesterol; and

wherein the first moiety is 90 to 100 molar percent and the second moiety is 0 to 10 molar percent of the sum of the total of the first and second moiety, wherein said nucleic acid is incorporated within the micellar or liposomal structures.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.